Rodriguez, batting with one out and a man on base, drilled a ball off a staircase above the wall in right-center field, good for a home run. But the ball bounced back into the field of play, and no umpire signaled for a homer.
Rodriguez slid into second before popping up to argue, and although the umpiring crew gathered to discuss the ruling, they did not overturn it.
"I was just running toward the ball and saw the ball kick back," Orioles right fielder Nick Markakis said. "I was just playing it like it was any regular ball hit and threw it back in. That's what the umpires are there for -- they're there to make that call."
Rodriguez, who hit a homer earlier in the game, has 524 career home runs.
"I didn't see it," Rodriguez said. "I could just tell by the reaction of all the guys in the dugout, and by the reaction of all the Baltimore guys, that it was a home run."
The play marked the second time in three days that a home run has been wrongfully taken away from a player at Yankee Stadium.
On Sunday night, Mets first baseman Carlos Delgado hit a ball off the base of the left-field foul pole, only to have umpires rule it a foul ball. The Yankees received the benefit of that call, and the detriment of this one.
Rodriguez did score later in the inning on Shelley Duncan's fielder's choice.
Mike Mussina, who often measures the clubhouse temperature by leaving notes on a white board next to his locker, did so again on Wednesday after the game.
"Balls that hit the yellow steps are usually homers," the white board read. "Not tonight."
Anthony DiComo is a reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.