Monopoly allegations against Microsoft go back several years
Microsoft has appealed against a 899m euros ($1.4bn; £680.9m) fine given for defying sanctions imposed on it for anti-competitive behaviour.
The penalty - the largest ever from the European Commission - came after it failed to comply with a 2004 ruling that it abused its market position.
The ruling said that Microsoft was guilty of not providing key information on interoperability to rivals.
Microsoft said it was appealing to seek "clarity from the court".
The Commission said that it was confident the fine was "legally sound".
Freezing out rivals
The challenge has been lodged with the EU Court of First Instance.
When they handed down the punishment in February, EU regulators said Microsoft was the first to break an EU anti-trust ruling.
March 2004: EU fines Microsoft 497m euros and orders it to release key Windows code to rival software developers
September 2004: Microsoft tries to have the ruling temporarily suspended
April 2006: Microsoft appeals against the ruling in the European Court of First Instance
September 2007: Microsoft loses its appeal
February 2008: EU imposes 899m euros fine on Microsoft for defying sanctions
May 2008: Microsoft appeals the fine, "seeking clarity"
The fines came on top of earlier fines of 280m euros imposed in July 2006, and of 497m euros in March 2004.
An investigation concluded in 2004 that Microsoft was guilty of freezing out rivals in products such as media players, while unfairly linking its Internet Explorer browser to its Windows operating system at the expense of rival browsers.
The European Court of First Instance upheld this ruling last year, which ordered Microsoft to pay 497m euros for abusing its dominant market position.
Earlier this year, Microsoft announced that it would open up the technology of some of its leading software, including Windows, to make it easier to operate with rivals' products.
The firm is still being pursued by Brussels.
Last month, the European Commission launched two new anti-competition investigations against Microsoft into similar issues.
The first will look at whether there are still problems regarding Microsoft's dominance of the PC software market.
The Commission will also investigate the continued interoperability of Microsoft software with rival products.