When Citrobacter freundii cephalosporinase was incubated with 6β-[3-(2-chlorophenyl)-5-methyl-4-isoxazolyl]penicillin sulfone (cloxacillin sulfone) in phosphate buffer, the enzyme was suddenly inactivated just after the completion of enzymatic degradation of the cloxacillin sulfone. Such delayed inactivation was due to a secondary inhibitor formed from cloxacillin sulfone during the incubation period. The inactivation was delayed due to the protection of the enzyme by cloxacillin sulfone from the attack of the secondary inhibitor. Phosphate anions were essential for the formation of the secondary inhibitor. However, once the secondary inhibitor was formed, the inactivation occurred in the absence of phosphate anions although the degree of the inactivation depended on the length of the preincubation period with phosphate anions. The main species (more than 80%) of the inactivated enzyme was detected as a single protein band with a slightly lower pI value than that of the native enzyme on isoelectric focusing on a plate.