The diastereomeric glucuronic acid conjugates are major metabolites of the nonsteroidal anti-inflammatory drug fenoprofen (FEN). Glucuronidation of FEN enantiomers was investigated with liver microsomal preparations from different species (sheep, rabbit, rat and human). The formed R- and S-FEN conjugates can be separated and quantitated directly on a C18 reversed-phase HPLC column using a mixture of acetonitrile and tetrabutylammonium sulfate buffer, pH 2.5, as mobile phase. Applying this analytical procedure, it is possible to characterize enantioselective glucuronidation of FEN. For in vitro procedures, rates of glucuronide formation are substrate (FEN) and cosubstrate (UDP glucuronic acid, UDPGA) dependent with initial rates of glucuronide formation being higher for R- than for 5-FEN. The R/S ratio of the formed products was independent of UDPGA (2.5–15 mmol/l) and substrate concentrations ≧ 0.4 mmol/l. Enantioselective cleavage of the formed FEN conjugates by alkaline hydrolysis and hydrolytic enzymes (R > S-glucuronide) can be controlled during in vitro studies by pH adjustment and the addition of enzyme inhibitors.