The disposition and immunogenicity of freshly prepared and stored solutions of benzylpenicillin (BP) and benzylpenicillenic acid (BPE), a degradation product of BP, were studied. No IgG anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA) following daily i.p. or i.m. administration to male Wistar rats of BP (2.7 mmol/kg) freshly dissolved in 0.5% glucose, for 4 consecutive days at 4-week intervals. In contrast, IgG anti-BPO antibodies were detected following both chronic i.p. and i.m. administration of BP (2.7 mmol/kg) stored for 24 hr at room temperature in 0.5% glucose. An IgG anti-BPO response was obtained only after the high dose, following daily i.m. administration of BPE (27μmol/kg, 2.7 μmol/kg, 0.24 μmol/kg). The specificity of the IgG antibody for the BPO-determinant was confirmed by ELISA inhibition with BPO-amino-caproate. Circulating BPO plasma-protein antigens were detected by a modified ELISA following i.p. and i.m. administration of both stored and fresh BP. Significantly lower BPO-antigen levels were detected in serum following BPE administration. Irreversible binding of BP to 75% rat plasma proteins was of the same magnitude when freshly dissolved in phosphate buffer or in 0.5% glucose (2.63 ± 0.32% and 2.55 ± 0.25% bound, respectively after 3 hr incubation at 37°). Irreversible binding was significantly greater (P < 0.05) when the BP was stored prior to incubation with the protein (3.81 ± 0.27%). The major degradation product of stored BP was benzylpenicilloic acid; a small amount of BPE (0.2% of incubated BP) was detected in stored but not fresh BP. Thus, the increased immunogenicity of BP stored for 24 hr at room temperature may be due to the formation of reactive degradation products such as BPE in vitro, which can then form immunogenic drug-protein conjugates in vivo. These experiments also show that although BP and BPE form drug-protein conjugates in vivo, circulating levels of antigen do not relate to the immunogenicity of either of the compounds.