The mechanism of synergistic activity of a combination of ampicillin and dicloxacillin was studied on β-lactamase-producing Citrobacter freundii GN 346 and its derived β-lactamaseless mutant GN346/16. The synergistic activity was exhibited against the parent strain but not against the mutant strain. Precultivation of the parent strain with the combination reduced the amount of the subsequent binding of [14C]penicillin G to the membrane fraction from the treated cells, but no reduction was observed in the case of cells treated with ampicillin or dicloxacillin alone. On the other hand, the amount of binding of [14C]penicillin G to the membrane fraction from the mutant strain was reduced by ampicillin treatment alone. These results cleary indicated that dicloxacillin inhibited the β-lactamase activity produced by the parent strain, and, consequently, ampicillin can penetrate through the outer membrane and periplasmic β-lactamase barrier into its target sites on the cytoplasmic membrane.