Furosemide (F) conjugation with glucuronic acid is the main pathway of F metabolism in humans and experimental animals. In order to study rat liver microsomal UDP-glucuronyltransferase (UDP-GT) activity towards F we developed an in vitro assay in which the conjugation product, furosemide 1-0-acyl glucuronide (FG) was separated and quantitatively determined by reverse phase high pressure liquid chromatography. The optimal conditions of the reaction were established and the apparent Km for F and UDP-glucuronic acid (UDPGA) were 0.22 and 1.76 mM, respectively. Substrate inhibition of UDP-GT toward F occurred at F concentrations higher than 1.5 mM. Developmental changes in F glucuronidation were compared to the ontogeny of UDP GT activity toward two other acceptors, 1-naphthol and estrone that are known to have different patterns of maturation. F glucuronidation was 26% of adult activity at 18 days of gestation, reached 48% at birth and gradually increased to 250% of adult activity at 22 days of age. Glucuronidation of 1-naphthol and estrone attained 87% and 44% of adult activity at 22 days of gestation, 37% and 66% in six-day-old rats and 100% and 427% of adult activity in 22-day-old rats, respectively. The effect of 3-methylcholanthrene (3-MC), phenobarbital (PB) and pregnenolone-16 alpha-carbonitrile (PCN) on F UDP-GT was studied and compared to their effect on 1-naphthol and estrone glucuronidation. PB, 3-MC and PCN increased F-UDP-GT activity to 208%, 282% and 342% of vehicle-treated animals, respectively, while F pretreatment did not affect the conjugation of F. In comparison, 1-naphthol glucuronidation was preferentially induced by 3-MC (4.4-fold of control) while estrone glucuronidation was induced by PB and PCN (4.9- and 2.5-fold of control, respectively). These studies suggest that several forms of UDP-GT activities, which differ in their ontogeny and inducibility patterns, are involved in the glucuronidation of F in vitro.