Penicillin V acylase from Bacillus sphaericus was purified to homogeneity with an overall yield of 15%. The enzyme exhibited comparatively high specificity for penicillin V, penicillin G, and other related compounds being hydrolyzed at less than 10% of the rate of penicillin V. Moreover, the high rate of hydrolysis was observed when the side chain of the substrate molecule was unsubstituted. Lysine-modifying reagents inactivated the enzyme rapidly. Kinetics and titration studies indicated the involvement of lysine in the catalytic activity of the enzyme.