UDP glycosyltransferases (UGTs) and sulfotransferases (SULTs) are phase II enzymes that interact with a number of xenobiotics in humans and animals. Species differences in enzymatic characteristics have seldom been investigated. Liver S9 fractions are commonly used for studying phase II metabolism in vitro. The objective of this study was to characterize the UGT and SULT activities in liver S9 fractions from various species including humans, monkeys, dogs, and rats. A single substrate, 7-hydroxycoumarin (7-HC), at several concentrations was incubated at 37° C with the S9 reaction matrices along with necessary cofactors. The rate of formation of two metabolites, 7-HC-glucuronide (7-HC-G) and 7-HC-sulfate (7-HC-S), was determined with Liquid Chromatography/Tanderm Mass Spectrometry (LC/MS/MS). Apparent K_m and V_max values were calculated for each species. For the UGTs, the apparent K_m and V_max for 7-HC-G formation varied greatly among different species, with dog UGTs having both the highest K_m and V_max values. In contrast to UGTs, the K_m for 7-HC-S formation showed no significant difference among humans, monkeys, and rats (approximately 3 μM). However, the K_m in dog was 8.7 μM. Species differences with respect to phase II metabolism must be carefully considered when selecting an in vitro model system to study various aspects of drug metabolism.