A study of the penicillin-binding proteins (PBPs) in the membranes of various species of Bacilli by dodecylsulphate/polyacrylamide gel electrophoresis followed by detection of the PBPs by fluorography has shown the presence of 3-5 such proteins depending on the species. Labelling of growing cells of Bacillus megaterium with [$^{14}C]benzylpenicillin showed that the PBP with the highest molecular weight, PBP1, was the only penicillin-binding protein that binds benzylpenicillin to any significant extent at the minimum growth inhibitory concentration. The covalent complexes formed between ~-lactams and the various penicillin-binding prdteins break down at different rates and the characteristics of the interaction of benzylpenicillin with PBP1 of B. megaterium are very similar to those of the inhibition by benzylpenicillin of the natural peptidoglycan transpeptidase reaction that is catalysed by cell-wall/ membrane preparations of this organism. PBP1 is proposed to be the peptidoglycan transpeptidase that represents the lethal target of penicillin action. Penicillin-binding proteins have been solubilized from the membranes with non-ionic detergents and isolated by affinity chromatography. Prior separation of the PBPs by ion-exchange chromatography followed by affinity chromatography has. resulted in the purification of PBPs 1, 3 and 4 of B. megaterium. PBP5 has also been purified by affinity chromatography and this purified protein catalyses a DD-carboxypeptidase reaction. None of the other purified PBPs has been shown to have enzymic activity in the terminal stages of peptidoglycan biosynthesis. The equivalent PBP1 from Bacillus licheniformis has been purified to ·protein homogeneity in large amounts in a single step process. Benzylpenicillin interacts with this purified protein to form a covalent stoicheiometric complex that subsequently breaks down and the half life of the complex is identical to that formed with the membrane-bound protein. The techniques used for the purification of PBP1 of B. megaterium have also been used to isolate PBP1 of Escherichia coli. The reactions that occur during the breakdown of benzylpenicillin-PBP complexes have been studied. The DD-carboxypeptidase of Bacillus stearothermophilus fragments benzylpenicillin to form phenylacetylglycine and N-formylD- penicillamine. The comparable reaction that occurs with PBP1 of B. licheniformis, either membrane-bound or in a purified form, appears to be more complex; bound benzylpenicillin is released via at least two different routes.