On the basis of a simple theoretical model, the ease of penetration of beta-lactam antibiotics through the outer membrane of Escherichia coli was measured. The cell envelope was found to act as a diffusion barrier to both penicillins and cephalosporins. The validity of the model and the cooperative action of cell-bound beta-lactamase and outer membrane were further verified by comparing calculated and experimentally determined velocities of beta-lactam hydrolysis by intact cells and sonically treated cell suspensions. The results showed good correspondence at five different antibiotic concentrations. Similar conclusions could be drawn from a comparison of beta-lactam concentrations on both sides of the outer membrane, calculated from enzyme kinetic measurements and minimal inhibitory concentrations for both a beta-lactamase-producing E. coli and its enzyme-negative variant. in the case of benzylpenicillin and cephalothin, however, no correspondence was found. The joint action of several parameters determining the efficacy of penicillins and cephalosporins against beta-lactamase-producing E. coli is discussed.