The format maximizes the chances for a slip-up by one of the top-tier teams, which usually play many backups and reserves in the early rounds.
Five of the MLS teams face USL-1 opposition, and in the cases of Houston, Chivas USA, and Kansas City the task is complicated by playing on the road.
The two-time MLS Cup champion Dynamo heads to Charleston (S.C.) to play the Battery at Blackbaud Stadium; Chivas USA heads up the coast to play Seattle at the Starfire Sports Complex in Tukwila, Wash.; and the Wizards face a tricky test at the WakeMed Soccer Park in Cary, N.C. against the Carolina Railhawks.
Seattle ended the Cinderella run of USASA club Hollywood United, which had stunned USL-1 Portland, 3-2, in the first round, but was pounded, 6-0, by the Sounders on Tuesday.
In the other USL-1/MLS matchups, D.C. United "hosts" Rochester at the Maryland SoccerPlex in Gaithersburg and FC Dallas plays Miami at its regular home, Pizza Hut Park.
Three MLS teams have been drawn against USL-2 teams. New England plays Richmond at Veterans Stadium in New Britain, Conn., and Chicago hosts the Cleveland City Stars, which went into overtime to upset USL-1 team Minnesota, 2-0, in the second round.
Red Bull New York plays Crystal Palace Baltimore, with the date and location yet to be announced. All other games are scheduled for Tuesday.
New England beat FC Dallas, 3-2, in the 2007 U.S. Open Cup final Oct. 3 at Pizza Hut Park. This year's schedule has pushed up the final to Aug. 26, with the semifinals set for Aug. 12 and the quarterfinals to be played July 8.
Prize money has been set as $100,000 to the champion, and $50,000 to the runner-up. Teams from Division II, Division III and amateur levels that advance farthest in the tournament will receive $10,000 each.