A large proportion of the metabolites formed from benzo[a]pyrene (BP) in cell cultures from rodents, fish and humans result from conjugation of an oxidized metabolite of BP with sulfate, glucuronic acid or glutathione (GSH). To improve the analysis of these metabolites, a reversed-phase ion-pair h.p.l.c. system using a step gradient of methanol:tetrabutyl-ammonium bromide in ammonium formate buffer has been developed for the separation of these three classes of conjugates. This system separated 3-hydroxy-BP glucuronide and sulfate conjugates and resolved them from GSH conjugates of BP 4,5-oxide, 7,8-oxide and 7,8-diol-9,10-epoxide. Cultures of early passage Syrian hamster, Wistar rat and Sencar mouse embryo cells, a bluegill fry (BF-2) cell line and a human hepatoma cell line (HepG2) were exposed to [3H]BP for 24 h. Medium samples from each were extracted with chloroform: methanol:water, and the water-soluble metabolites were analyzed by ion-pair h.p.l.c. The largest peak of metabolites in the media from cell cultures from rodents and the bluegill fry cell line co-eluted with the glucuronic acid conjugate of 3-hydroxy-BP. These phenol-glucuronides represented 48-62% of the total water-soluble metabolites in the fish and rodent cell cultures. Treatment of this material with beta-glucuronidase released 3-hydroxy-BP and 9-hydroxy-BP in ratios from 3:4 to 13.3:1 in various cultures. Media from the bluegill fry cell line and the mouse embryo cell cultures also contained a peak of BP-diol glucuronides; treatment of these peaks with beta-glucuronidase released mainly BP-7,8-diol. In HepG2 cells, 40% of the water-soluble metabolites were identified as sulfate conjugates of 3-hydroxy-BP and 9-hydroxy-BP. No glucuronic acid conjugates of BP metabolites were detected in HepG2 cells. Only small amounts of the water-soluble metabolites from these cell cultures eluted in the same volumes as the synthetic GSH conjugate of BP-4,5-oxide, BP-7,8-oxide and BP-7,8-diol-9,10-oxide. These studies indicate that conjugation with glucuronic acid represents a major pathway of formation of water-soluble metabolites from BP in cells derived from a number of species and demonstrate the value of this ion-pair h.p.l.c. system for the analysis of conjugates formed from BP.