The mode of bacterial killing by penicillins is still unknown in spite of many studies on the subject. The recent finding of multiple penicillin binding proteins (PBPs) in sensitive bacteria and the possibility of analysing the binding of the antibiotic to exponentially growing cells have provided new directions for investigating this problem. Sensitivity to lethal and other effects of penicillin varies very significantly with the conditions of growth of the cells. If PBPs were the penicillin target, changes in conditions of growth causing variations in penicillin sensitivity should be accompanied by changes in these proteins. Furthermore, if one of PBPs could be identified as the killing target, it could possibly be demonstrated to show changes in cells growing in different conditions. We show here that in Streptococcus faecalis ATCC 9790 changes in conditions of growth are accompanied by changes in PBPs. Furthermore, in the presence of the minimal dose of 14C-benzylpenicillin causing complete inhibition of cell growth, 100% of the total radioactivity is bound to a single protein (PBP 3).