The degree to which cell-wall-deficient bacteria (CWDB) are involved in the generation of beta-lactamase derepressed mutants (DM) was measured using Enterobacter cloacae 3624. The frequency of DM in non-permissive isotonic ticarcillin medium was compared with their frequency in hypertonic ticarcillin medium that supports CWDB growth. DM were resistant to extended spectrum penicillins and cephalosporins and had a basal beta-lactamase activity of >300 units/mg protein. Anaerobic growth of CWDB increased the relative risk of DM 2 x 10(6)-fold. Aerobic incubation produced fewer CWDB colonies but the risk of DM was still increased 400-fold over non-permissive controls. These results define a new role for CWDB as intermediaries in the emergence of resistance.