More than 130 businesses have signed a letter saying the business case for Scottish independence “has not been made”.
The signatories come from a variety of businesses including banking, mining, engineering, food, whisky, and technology.
But lobby group Business for Scotland said economic “facts and figures” support Scottish independence.
The letter has been published in The Scotsman newspaper.
Scotland will go to the polls in the referendum on 18 September, with voters being asked the “Yes/No” question: “Should Scotland be an independent country?”
The letter is signed by business leaders including Douglas Flint, the chairman of HSBC; Andrew Mackenzie, the chief executive of the mining giant, BHP Billiton; and Sir John Grant, executive vice-president of policy at the energy company, BG Group.
Others include Audrey Baxter, executive chairman of Baxters Food Group; Simon Thomson, the chief executive of Cairn Energy; and Ian Curle, the chief executive of Edrington, which owns the whisky brands The Macallan and The Famous Grouse.
The letter was organised by Keith Cochrane, the chief executive of the engineering firm, Weir Group, who has already warned of “substantial risks” if Scotland votes for independence. Its backers say that no political organisations have been involved in putting together the list.
The letter says that “much is at stake”: “Our economic ties inside the United Kingdom are very close and support more than a million Scottish jobs.
“As job creators we have looked carefully at the arguments made by both sides of the debate.
“Our conclusion is that the business case for independence has not been made.”
One source told BBC Business Editor Kamal Ahmed that it grew out of a “frustration” about what is perceived as a lack of cohesiveness about the business and independence debate.
The signatories account for about 50,000 jobs in Scotland, from large firms such as Babcock ship building to small engineering firms in Glasgow’s east end, our business editor says in his blog.
Supporters of an independent Scotland point to their own supporters from business and economics.
Business for Scotland said in a statement: “Business for Scotland has 2,500 members who run businesses in Scotland, employ people across the country in a range of industries, and all believe that Scottish independence is in the best interests of Scotland and Scottish business.
“It’s a position reached after looking at the facts and figures and realising that, from a simple balance sheet point of view as well as other considerations, our best interests lie in becoming an independent country.”
Former Royal Bank of Scotland chairman Sir George Mathewson, has said that Scotland’s vital financial services sector – for example – would flourish if Scotland votes “Yes”.
“There is nothing to suggest that being part of a smaller country hinders a financial services industry. Switzerland, for example, has – in Geneva and Zurich – not one, but two of the world’s Top 10 financial centres. Singapore, with five million people, is ranked 4th,” he has said.
“Investment is an increasingly global business, where success depends much more on people than on borders.”
Economist and former head of Scottish Enterprise Sir Donald MacKay has argued that an independent Scotland would be in better fiscal shape than the UK is at present.
“An independent Scotland should use that financial advantage to invest in re-engineering our economy towards industrial, manufacturing and tradeable services development,” he has said.
Willie Walsh, the chief executive of IAG, owner of British Airways, also says that independence could be good for Scotland.
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