About 70%o of all Enterobacter cloacae strains tested possessed one of two species-specific 3-lactamases. These enzymes, E. cloacae ,B-lactamase A and E. cloacae ,-lactamase B, with isoelectric points of 8.8 and 7.8, respectively, had the same pH and temperature optima. Both showed similar enzyme kinetics and were inhibited by cloxacillin but not by p-chloromercuribenzoate. E. cloacae 3-lactamase B appeared to be identical with the enzyme of E. cloacae P99. By a mutation in a regulatory gene, inducible enzyme production could be converted into constitutive expression. In E. cloacae, both enzymes did not hydrolyze thirdgeneration cephalosporins, but they were solely responsible for resistance toward these drugs. This was demonstrated by the characterization of Escherichia coli strains expressing an identical resistance pattern after transfer of the corresponding Enterobacter gene.The importance of plasmid-coded 1-lactamases for the resistance of gram-negative bacteria to ,-lactam antibiotics is well established (6-8, 12, 17, 21, 24). In contrast, the contribution of chromosomal lactamases to resistance remains unclear. One exception is Escherichia coli mutants in which constitutive expression of the chromosomal ampC gene contributes to ampicillin resistance (2,3). Recently, however, evidence has begun to accumulate that in certain Enterobacter cloacae strains, the chromosomal ,B-lactamases may play an unexpected role (1, 13,22,23,25,28). These enzymes are typical cephalosporinases which usually can be induced by penicillins and cephalosporins (4,22,23 (25) proposed that thirdgeneration cephalosporins are not inactivated by hydrolysis but by a kind of trapping mechanism, or that other intrinsic factors (13,28) are involved in this process.In this paper we give evidence that (i) there are mainly two chromosomal ,3-lactamases in E.cloacae strains, (ii) that resistance to thirdgeneration cephalosporins arises by mutation to constitutive production of the P-lactamases, and (iii) that these P-lactamases are solely responsible for the resistance. In addition, we provide enzymatic data to characterize these enzymes.MATERIAULS AND METHODS Bacteril sra. A total of 61 cefotaxime-susceptible E. cloacae strains, isolated in 1979 from clinical specimens, were used for the epidemiological study. Five of these strains (E. cloacae 314, 352, 150, 988, and 149)