Allantoin catabolism studies have been extended to intact leaf tissue of soybean (Glycine max L. Merr.). Phenyl phosphordiamidate, one of the most potent urease inhibitors known, does not inhibit (14)CO(2) release from [2,7-(14)C]allantoin (urea labeled), but inhibits urea dependent CO(2) release >/=99.9% under similar conditions. Furthermore, (14)CO(2) and [(14)C] allantoate are the only detectable products of [2,7-(14)C]allantoin catabolism. Neither urea nor any other product were detected by analysis on HPLC organic acid or organic base columns although urea and all commercially available metabolites that have been implicated in allantoin and glyoxylate metabolism can be resolved by a combination of these two columns. In contrast, when allantoin was labeled in the two central, nonureido carbons ([4,5-(14)C]allantoin), its catabolism to [(14)C]allantoate, (14)CO(2), [(14)C]glyoxylate, [(14)C]glycine, and [(14)C]serine in leaf discs could be detected. These data are fully consistent with the metabolism of allantoate by two amidohydrolase reactions (neither of which is urease) that occur at similar rates to release glyoxylate, which in turn is metabolized via the photorespiratory pathway. This is the first evidence that allantoate is metabolized without urease action to NH(4) (+) and CO(2) and that carbons 4 and 5 enter the photorespiratory pathway.