Millions of subscribers lost files hosted with the service when the US authorities shut down the website in January for alleged copyright violations.
Film studios are expected to argue that any illegally gathered data should remain locked up as evidence, but the first order of business could be whether Megaupload's lawyers are allowed to address the court, according to CNET.
Neil MacBride, the US attorney who indicted Megaupload co-founder Kim Dotcom, claims that the internet company's legal representation Quinn Emanuel should not be able to make an address without the presence of the defendants.
Moreover, MacBride argues that there is a conflict of interest because one of Quinn Emanuel's lawyers represented YouTube in a past dispute. The charges against Megaupload include unlawfully copying YouTube content.
Digital rights groups are campaigning to have legitimate data store on Megaupload's servers returned to users. The Electronic Frontier Foundation will argue the case on behalf of service user Kyle Goodwin, who used the cloud locker to back up files for his business.
Goodwin, who earns a living filming high school sports events in Ohio, lost material imperative to his work during a hard drive failure, and is demanding access to the back-ups.
Megaupload's servers are stored in a facility run by Carpathia. The hosting company, which is also pressing for a resolution to the data issue, shut down the servers, claiming it was costing $9,000 (£5,700) to keep them running.
The Virginia court will begin debating the issue of Megaupload user data today at 9am local time (2pm GMT).