Gentisamide (2,5-dihydroxybenzamide, GAM), a substrate that forms two monosulfates at the 2 and 5 positions (GAM-2S and GAM-5S) and a monoglucuronide at the 5 position (GAM-5G), was delivered at 8 or 80 microM by normal (N) and retrograde (R) flows to the once-through rat liver preparation. At the lower (8 microM) input concentration, ratios of conjugate formation rate, GAM-5S/GAM-5G and GAM-2S/GAM-5G, were decreased significantly (4.01 +/- 1.42 to 2.93 +/- 0.99, and 1.13 +/- 0.65 to 0.66 +/- 0.41, respectively) whereas a small but significant increase in the steady-state extraction ratio, E (0.89 +/- 0.029 to 0.94 +/- 0.016), was observed upon changing the flow direction from N to R. At the higher input GAM concentration (80 microM), conjugate formation rate ratios were relatively constant for GAM-5S/GAM-5G (1.18 +/- 0.08 to 1.11 +/- 0.12) and GAM-2S/GAM-5G (0.33 +/- 0.05 to 0.31 +/- 0.05) upon changing flow direction from N to R, despite a slight increase in E from 0.87 +/- 0.023 to 0.91 +/- 0.016 was observed. These results suggest that the sulfotransferase activities responsible for 2- and 5-sulfoconjugations are identically distributed and localized anterior to 5-glucuronidation activities during a normal flow of substrate into the rat liver (entering the portal vein and exiting the hepatic vein), and that the presence of uneven distribution of conjugation activities is discriminated only at the lower input drug concentration. At high concentration (greater than Km for all systems), saturation of all pathways occurs, and other anteriorly/identically distributed competing pathways would fail to perturb downstream intrahepatic drug concentrations and the resultant conjugation rates. The lack of change in metabolic profiles renders the condition unsuitable for examination of uneven distribution of enzymes in the liver. These observations were generally predicted by theoretical enzymic models of consistent distribution patterns. Because 2- and 5-sulfation were mediated by systems of similar Km but different Vmax values, two possibilities, the same isozyme of sulfotransferase being involved in the formation of two enzyme-substrate complexes to form two distinctly different products or two isozymes of sulfotransferases of identical distribution, were discussed.