Strains of Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae that were resistant to ceftazidime (MIC > 16 ,ug/ml) but susceptible to BMY 28142 (MIC < 4 ,ug/ml) were found to contain higher levels of j-lactamase activity (50-to 3,340-fold) than control strains of the corresponding species. Ceftazidime was at least as resistant as BMY 28142 to hydrolysis by these enzymes. However, the apparent K, of BMY 28142 for each enzyme was larger (8-to >20-fold) than that of ceftazidime; i.e., the affinity of these enzymes for BMY 28142 appeared to be lower than that for ceftazidime. Thus, BMY 28142 was affected less than ceftazidime by a mechanism of resistance that depends, at least in part, on the relative affinities of cephalosporins for the j3-lactamases of these species. These results indicate that the affinity between a jI-lactamase and a cephalosporin may be a distinguishing factor in the evaluation of ,-lactamase-resistant cephalosporins and suggest that affinity can play a major role in susceptibility to highly ,(-lactamase-resistant cephalosporins.During routine evaluation of a new cephalosporin, BMY 28142 (5), occasional clinical isolates of Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae, as well as the P-99 strain of E. cloacae, were found to be resistant (MIC > 16 pug/ml) to ceftazidime but susceptible to BMY 28142 (MIC < 4 ,ug/ml). Resistance to cephalosporins that are intrinsically stable to P-lactamase hydrolysis has been correlated with high levels of ,B-lactamase activity (1, 2, 9-11, 14). The concept of trapping or nonhydrolytic interference was proposed to explain this apparently paradoxical phenomenon (8,10,11,14,15). Criticism of this concept, based on the premise that for this mechanism to operate the total number of P-lactamase molecules must exceed the number of P-lactam molecules (9), does not take into consideration the presence of the outer membrane barrier, as discussed by Vu and Nikaido (13). Their analysis, based on outer membrane permeability, kinetic parameters of the enzyme, affinity toward penicillin-binding proteins, and MIC, suggests that hydrolysis may be more important than nonhydrolytic binding for ,-lactams such as cefoperazone, cefoxitin, and cefotaxime (13). However, the results of their calculations for ceftazidime, which is much more resistant to hydrolysis than the other three cephalosporins, were less conclusive with respect to hydrolysis versus nonhydrolytic interference as the major determinant of resistance in the single E. cloacae strain that they examined.We therefore investigated the contribution of P-lactamaserelated factors to the difference in the relative activity of ceftazidime and BMY 28142 against some of these ceftazidime-resistant isolates. For each species one to three strains resistant to ceftazidime but susceptible to BMY 28142 were compared with a control strain for which MICs of BMY 28142 and ceftazidime were similar to the respective MIC50s * Corresponding author. t Present address: