The assay of UDPglucuronosyltransferase activity toward various substrates using UDP[U-14C]glucuronic acid is described. HPLC on a polar amino-cyano bonded phase column was used to separate radioactive glucuronides from unmetabolized UDP[U-14C]glucuronic acid and other labeled reaction products. Radioactivity was measured using flow-through scintillation counting. All the glucuronides analyzed, with one exception, chromatographed with the same retention time (9.0-9.6 min) under the conditions described. Glucuronide conjugates were identified by comparison with retention times of commercial glucuronide standards, using radioactive aglycones, or hydrolysis with beta-glucuronidase. The method provides a unified, sensitive (100-200 pmol of glucuronide product) and reproducible assay for a wide variety of UDPglucuronosyltransferase substrates, and could be extended to include many others.