Chromatography of benzo[a]pyrene (BaP) sulfate, glucuronide and glutathione (GSH) conjugate standards were examined by h.p.l.c. on a C8 column as modified by various organic acids and solvents. Sulfate and glucuronide standards were positional isomers derived from BaP-1,3,6,7,9 phenols and BaP-GSH conjugates consisted of a racemic mixture of BaP-4,5-GSH. In the absence of acid, BaP conjugates appeared as rapidly eluting, unresolved peaks in aqueous-methanol or acetonitrile gradients or coeluted as broad peaks in a water-propanol gradient, with the exception of BaP-7-OH sulfate which eluted as a distinct symmetrical peak. Addition of acetic or trifluoroacetic (TFA) acids enhanced column retention of BaP conjugates in each solvent system. Upon acidification of mobile phases, BaP-GSH isomers were partially resolved, isomers of BaP sulfates or of BaP glucuronides coeluted, and BaP-7-OH sulfate was resolved from all conjugates. BaP-GSH conjugates were most resolved and preceded elution of other conjugates when TFA was added to mobile phases. BaP sulfates and glucuronides generally coeluted but were partially resolved at 0.1% TFA in a water-methanol gradient. Water-soluble metabolites from cultured hamster embryo fibroblasts (HEF) incubated with [3H]BaP for 24 h were chromatographed by h.p.l.c. in a water-methanol gradient with TFA. BaP glucuronides, consisting of tetraols, triols, quinones, dihydrodiols and phenols eluted as a single peak which could be removed by beta-glucuronidase treatment and organic extraction. BaP sulfates were not detected. The remaining BaP metabolites which were resistant to enzymatic hydrolysis, generally eluted prior to BaP glucuronides suggesting they constitute a family of BaP-GSH derivatives.