The hydrolysis of 6-aminopenicillanic acid (APA) at neutral pH, catalyzed by the class C beta-lactamase of Enterobacter cloacae P99, was observed from H-1 NMR spectra to yield an unexpected product, 8-hydroxypenillic acid, in bicarbonate-containing buffers. This product probably arises from turnover of the carbamate of APA by the enzyme. H-1 and C-13 NMR spectra of APA in bicarbonate solutions clearly demonstrate the presence of the carbamate. Turnover of APA at saturating concentrations by the enzyme is accelerated by bicarbonate and by methanol. These results suggest that deacylation of the enzyme is rate determining and that the presence of the carbamate affects this step. A mechanism involving rate-determining intramolecular nucleophilic attack by the carbamate on the acyl enzyme is proposed to rationalize these observations and lead to formation of 8-hydroxypenillate. This reaction can be seen as an example of substrate-assisted enzymic catalysis. Quantitative analysis of the data indicated a dissociation constant of the APA-carbamate to APA and bicarbonate of 7 mM at pH 7.5. The P99 beta-lactamase also catalyzes the formation of an alternative product from 7-aminocephalosporanic acid in the presence of bicarbonate. Ampicillin and cephaloglycine which also possess an amine-bearing side chain [PhCH(NH2)CO-] react with bicarbonate to form carbamates at neutral pH but these do not yield alternative products on turnover by the P99 beta-lactamase. Typical class A beta-lactamases do not catalyze 8-hydroxypenillate formation from APA and bicarbonate even when deacylation is rate determining. This difference is discussed in terms of beta-lactamase active site structure.