Decreased susceptibilities to two low Mr beta-lactam antibiotics, CS-533 (a carbapenem; Mr, 339) and CGP31608 (a penem; Mr, 262), were found in imipenem-resistant mutants of Pseudomonas aeruginosa PAO. The diffusion rates of several beta-lactam antibiotics including imipenem, CS-533 and CGP31608 across the proteoliposome membrane reconstituted from the outer membrane of the wild type strain or its imipenem-resistant mutants were determined by the liposome swelling technique. Diffusion rates of imipenem, CS-533 and CGP31608 in the proteoliposomes from outer membranes of the imipenem-resistant strain were found to be 27, 20 and 47%, respectively, of the diffusion rates in proteoliposomes reconstituted from outer membranes of the sensitive parent strain. The SDS-polyacrylamide gel electrophoretogram of outer membrane proteins of the imipenem-resistant strains indicated deletion of protein D2. These results suggested that decreased susceptibilities to imipenem, CS-533 and CGP31608 were due to decreased outer membrane permeability, and that D2 is a protein fraction constituting pores for diffusion of these antibiotics through the P. aeruginosa outer membrane.