A method was developed which permitted determination of the [14C]benzylpenicillin and [14C]Cephapirin binding capacity of rapidly growing Bacillus subtilis cells in liquid culture. Over the concentration range of the binding plateau (0.1 to 0.8 mug/ml), [14C]benzylpenicillin significantly inhibited formation of functional penicillin-binding proteins, but had comparatively little effect on total bacterial protein synthesis. The data suggest that penicillin covalently bound to the cells in a chemically stable manner alone is not sufficient to inhibit formation of functional binding proteins and that unbound penicillin in the growth medium is necessary. The concentration of unbound antibiotic in the culture medium, in turn, is a function of the cell-bound penicillinase activity whose significance increases with cell density. [14C]Cephapirin, a cephalosporin resistant to this cell-bound penicillinase almost completely inhibited the formation of functional Cephapirin-binding proteins, but had relatively little effect on total protein synthesis. At concentrations 250-fold higher than that required to inhibit formation of functional binding proteins. Cephapirin did not inhibit particulate D-alanine carboxypeptidase activity and presumably did not bind covalently to this penicillin-binding protein.