Sulfation of phenols and similar low-molecular-weight substrates in the rat in vivo is a rather complex process. Besides enzyme kinetic parameters, cosubstrate availability (indirectly measured by serum sulfate concentration) and competition with glucuronidation also play a role. For some substrates extensive extrahepatic sulfation occurs, accounting for more than 50% of the total-body sulfation capacity. However, the hepatic contribution may be under-estimated when drugs are administered into the hepatic portal vein, because saturation of hepatic metabolism may occur under those conditions. Inside the liver, sulfation is located primarily in zone 1, the periportal area. This can be shown in the single-pass perfused rat liver by perfusion in either the normal or retrograde flow direction. In the rat sulfate conjugates are eliminated preferentially in urine, whereas glucuronides are excreted to a high extent in bile. Therefore, it is important to collect both bile and urine in the characterization of pharmacokinetics of conjugation in vivo. Selective inhibition of sulfation by pentachlorophenol and 2,6-dichloro-4-nitrophenol facilitates studies of the role of sulfation in elimination of its substrates, and the competition between sulfation and glucuronidation for the same substrate.