Abraham & Newton (1956) showed that cephalosporin C was highly resistant to hydrolysis by purified penicillinase from BaciUu8 cereus (strain NRRL 569; see Pollock, 1959, 1960) but was a competitive inhibitor of the action of this penicillinase on benzylpenicillin. More recently, however, cephalosporin C was reported to have no significant inhibitory action on the hydrolysis of benzylpenicillin by penicillinase from a strain of Staphylococcus aureus (Abraham & Newton, 1961 a). Rolinson, Stevens, Batchelor, Wood & Chain (1960) reported that 2,6-dimethoxyphenylpenicillin was a competitive inhibitor of penicillinase from B. cereus, but not of penicillinase from Staph. aureus. These findings suggested that penicillinase from B. cereus and penicillinase from Staph. aureus differed with respect to structural features which influenced the combination of enzyme and substrate. They also drew attention to the fact that the affinity for penicillinase of a member of the cephalosporin C or penicillin family might partly determine the rate of enzymic hydrolysis of the substance in the concentration at which it was used as an antibacterial agent. The results of further experiments in this field are given here. The substances studied were N-acyl derivatives of 7-aminocephalosporanic acid which has structure (I; R = H, R' = CH3 Co00)