r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BaP-tetraol) is the major hydrolysis product of r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the principal ultimate carcinogen of the environmental pollutant benzo[a]pyrene (BaP). As part of a program to establish activation/detoxification profiles of urinary metabolites of BaP in humans, we developed a method for quantifying trans-anti-BaP-tetraol. Urine was collected from three groups of individuals exposed to BaP: psoriasis patients treated with a coal tar-containing ointment, steel workers, and smokers. [(2)H(12)]-trans-anti-BaP-tetraol was added to the urine as an internal standard. The urine was treated with beta-glucuronidase and sulfatase, and then the BaP-tetraols were enriched by reverse-phase and phenylboronic acid solid-phase extraction. The resulting fraction was treated with sodium hydride and methylmethane sulfonate to convert BaP-tetraols to the corresponding tetramethyl ethers (BaP-TME). The mixture was purified by normal-phase HPLC and analyzed by gas chromatography/negative ion chemical ionization/mass spectrometry with selected ion monitoring. [(13)CH(3)](4)-trans-anti-BaP-TME was used as an external standard. Ions at m/z 376, 380, and 388 were monitored for quantitation of trans-anti-BaP-TME, [(13)CH(3)](4)-trans-anti-BaP-TME, and [(2)H(12)]-trans-anti-BaP-TME, respectively. The instrumental detection limit was approximately 1 fmol of trans-anti-BaP-TME. trans-anti-BaP-tetraol (as trans-anti-BaP-TME) was detected in 20 of 20 individuals receiving coal tar therapy (mean, 16 fmol/mL of urine), 13 of 13 exposed steel workers (mean, 4.1 fmol/mL of urine), and nine of 21 cigarette smokers (mean, 0.5 fmol/mL of urine). The means in these groups were significantly different (P < 0.0001). The urine of steel workers was also analyzed for cis-anti-BaP-tetraol and cys-syn-BaP-tetraol, but neither was found. The results of this study provide a quantitative method for determination of parts per trillion levels of trans-anti-BaP-tetraol in human urine. Ultimately, this method can be employed as part of a phenotyping approach for assessing BaP metabolites in human urine.