The degree of peptidoglycan cross-linking has been studied in growing cells of a Dap(-) Lys(-) auxotroph of Escherichia coli K-12 by following the incorporation of [(3)H]diaminopimelic acid into the lysozyme digestion products of crude, isolated peptidoglycan. The percentage of inhibition of cross-linking increases with increasing concentrations of penicillin G, cephaloridine, and cefuroxime. When the R factor R1drd 19 was introduced into the strain by conjugation, it was found that the type IIIa, beta-lactamase specified by the plasmid was able to protect the cross-linking target against inhibition by penicillin G but not against cephaloridine, even though the beta-lactamase hydrolyzes this substrate 50% faster than penicillin G. Cefuroxime, which is completely resistant to hydrolysis by the type IIIa beta-lactamase, inhibited the peptidoglycan cross-linking target in both the R(+) and R(-) variants of the assay strain. A mutant plasmid, R1drd19amp2, which specified no type IIIa beta-lactamase synthesis, could not provide protection of the cross-linking target against penicillin G. The significance of these results, in relation to the ability of the antibiotics to pass the permeability barrier of the bacterial envelope, is discussed.