The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.