The stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45 degrees C. It increased with the substrate (k(cat)/K-m) and nucleophile (k(T)/k(H)) specificity, and was found to differ in the S-1- (R-specific) and S-1-(S-specific)-binding subsites of the active site. The S-1-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.