Quantitative determination of the hydrolysis products from proteins and DNA gives valuable information regarding the reactive metabolite that forms the protein and DNA adduct. Quantification of protein-benzo[a]pyrene (BP) adducts represents a more sensitive method than quantification of BP-DNA adducts. The aim of the present study was to identify two hydrolysis products from BP-derived protein adducts found in vitro and in vivo in a previous study. Male Wistar rats were injected i.p. with BP, and serum albumin was isolated and subjected to acid hydrolysis at 70 degrees C for 3 h. The hydrolysate was subjected to LC separation, and fractions of the two unknown compounds were collected. The molecular masses of the two unknown compounds were in accordance with being tetrols as judged by LC electrospray mass spectrometry. The fragmentation patterns were characteristic of tetrols with formation of the molecular ion and the loss of water molecules. In addition, the compounds were subjected to acid hydrolysis at 70 degrees C with 0.1 M HCl for 3 h. We observed that two of the known tetrols epimerized to the two unknown tetrols and vice versa. This is probably a characteristic epimerization involving not only position C(10)-OH but also another site like position C(7)-OH. The in vivo findings of the two unknown adducts are probably the result of the formation of BPDE III in the metabolism of BP. These two tetrols must then have the C(7)-OH and C(8)-OH groups in a cis position.