The disposition and metabolic fate of benzylpenicillin conjugated to a protein, human serum albumin (HSA), were compared with those of free penicillin in the rat. The conjugate was prepared by in vitro incubation of [3H]-benzylpenicillin and HSA at pH 10.8 for 24 hr at 37 degrees, conditions which favour the formation of penicilloyl-lysine residues. The synthetic conjugate was cleared more slowly from plasma than free penicillin after intravenous administration; thus at 3 hr, concentrations of 5.08 +/- 0.50% dose/ml of the conjugate (0.31 microCi; 2.92 mg protein) was obtained. In an earlier study a concentration of 0.03 +/- 0.01% dose/ml was obtained after administration of free BP (2.7 mmol kg-1). During this time, 1.41 +/- 0.50% of the conjugate dose was excreted in urine while 5.0 +/- 0.2% of the dose was excreted in bile. Tissue analysis indicated that the liver contained 15.3 +/- 0.9% of the dose, while other tissues contained less than 6% of the dose. In long term metabolism studies it was found that 39.5 +/- 1.0% and 46.5 +/- 0.9% of the dose (0.43 microCi; 6.33 mg protein) was excreted in the urine after 3 and 7 days respectively. The principal metabolite (63-68%) excreted in both bile and urine was identified on the basis of cochromatography and fast atom bombardment mass spectrometry as benzylpenicilloic acid, indicating that the conjugate undergoes specific cleavage at the bond between the benzylpenicilloyl moiety and the protein. In vitro degradation studies indicate that the metabolism occurs primarily in the liver. Therefore benzylpenicilloic acid excreted in urine, after administration of free BP, may be formed either by direct hydrolysis of the beta-lactam ring, and/or result from catabolism of protein conjugates formed in vivo.