Football Club West Ham United, Selfridges and the London School of Economics, have become some of the first owners of a .London domain.
Other brands signing up for new web address include the London Eye and the Meantime brewery.
From Wednesday, companies in the capital are able to apply for the domain as an alternative to the .co.uk or .com addresses.
Global internet body Icann is introducing more than 1,000 new web addresses, seen as one of the biggest changes online since the invention of the World Wide Web.
While several major cities will have their own domain names, London is the first to announce its initial users.
The first sites go live on April 29. London-based firms will have a three-month preference window in which to purchase a .london domain.
Mayor Boris Johnson said: “There is enormous interest in .london from businesses right across the capital, not just from high street brands but from the small businesses that are the lifeblood of London’s economy.
“London leads the world in technology and our businesses are among the most dynamic and innovative anywhere so it is no surprise that so many see .london for the great opportunity it presents them.”
Karren Brady, vice-chairman of West Ham, said: “As the traditional London club, with roots formed at the heart of the East End in 1895, a move to become the first football club to use the .london domain is a natural fit for West Ham United.”
The new domain was officially launched at Covent Garden Market, with businesswoman and Dragons’ Den star Deborah Meaden offering her support for the scheme.
As part of the launch, the 40 businesses leading the scheme have written an open letter calling on more companies to follow suit and support the new domain name as a way of boosting the economy in London.