An HPLC method has been developed for the determination of the cephalosporin antibiotic Ro 13-9904 in plasma, urine, and bile of dogs and of human volunteers using the technique of ionpairchromatographywith a LiChrosorbRP-18 column.The three mobile phases employed contained tetrapentyl-, tetraoctyl-and hexadecyltrimethyl-ammonium bromide, respectively, as lipophilic counterions. The chromatographic conditions chosen allowed simple and rapid sample preparation. Plasma was deproteinired with ethanol and the supernatant was directly injected onto the column; urine and bile were diluted with mobile phase and injected without any purification. The detection limit for the cephalosporin was about 0.5 pg/ml for plasma samples and approximately 5 pg/ml for bile and urine.