An isogenic set of mutants of Pseudomonas aeruginosa, altered in permeability or permeability plus constitutive production of beta-lactamase, was examined for susceptibility to newer beta-lactam antibiotics. Kinetic data on the chromosomal beta-lactamase and susceptibility studies for the test beta-lactams indicate that permeability was the major mechanism of resistance to the poorly hydrolyzed and nonhydrolyzed antibiotics, e.g., carbenicillin, moxalactam, and cefsulodin. An exception was cefotaxime, with a low Km and a low Vmax, which had reduced efficacy in the permeability mutant and was further affected by the constitutive beta-lactamase. In this case, since the Vmax was low, a nonhydrolytic barrier may provide the additional reduction in susceptibility.