Zambrano (3-2) gave up four runs before he was pulled. He will be examined again on Sunday.
"It's like a strain in the forearm muscle," Cubs manager Dusty Baker said. "He didn't feel it in the last game, he didn't feel it on the side. In the fourth inning, that's the first time he felt it."
Baker said he didn't think Zambrano was affected by the change in starting time on Saturday. The game was delayed two hours and 40 minutes because of rain, although that didn't seem to dampen the enthusiasm of the crowd of 42,829, the second largest at RFK Stadium.
"I don't think it had anything to do with the conditions," Baker said.
The right-hander was making his first start since his 136-pitch complete-game victory against Philadelphia on May 8, but he'd had an extra day of rest.
"He's sore on the lateral aspect of his elbow," Cubs athletic trainer Mark O'Neal said. "It's more commonly known as tennis elbow.
"I asked him specifically if he felt it between his last start, in the middle, on the side, in the first three innings tonight. He denied that," O'Neal said. "It just came on. It looked like he was stretching his forearm a little bit and that's when we went out there. He denied it initially on the field. Then he let us know it bothered him and we got him out of there."
The problem areas are the muscles that extend up the wrist and the low end of his biceps, O'Neal said. Asked if the injury was minor, O'Neal said, "We'll treat it as something we really want to watch.
"To use the terms of the doctors here, if you're a pitcher and have elbow pain, this is the site where you want to have elbow pain on," O'Neal said.
Zambrano retired the first nine batters he faced Saturday before Brad Wilkerson doubled to lead off the Nationals' fourth.
Then it got messy. Wilkerson was safe at third on Jamey Carroll's fielder's choice as Zambrano's throw to third pulled Aramis Ramirez off the bag. One out later, Zambrano hit Nick Johnson to load the bases. Castilla then hit a two-run single, with both runners advancing on left fielder Jason Dubois' wild throw home. Ryan Church added an RBI single, Brian Schneider was hit by a pitch and Cristian Guzman hit an RBI single.
O'Neal sprinted to the mound after Zambrano threw the third pitch to Nationals starter Livan Hernandez, and Zambrano was pulled. He had thrown 64 pitches.
Last year, Zambrano left the July 2 game against the Chicago White Sox because of cramps in his right forearm, but that was because of dehydration.
Zambrano helped the Cubs take a 1-0 lead in the third when he hit a two-out double and scored on Corey Patterson's ground-rule double. Pinch-hitter Jose Macias hit an RBI single in the Chicago fifth to pull within 4-2.
Hernandez (6-2) gave up two runs on five hits over 6 1/3 innings, and struck out six for the win. However, he had to leave the game because of inflammation in his right knee.
The Cubs rallied in the ninth, and added a run on Corey Patterson's RBI single.
"Our guys battled back and we had action," Baker said. "It's hard coming up short like this."