1. The metabolism of D,L-rolipram in man was studied by comparison of h.p.l.c. radiochromatograms of plasma and urine obtained at various times following oral and/or s.c. administration to man, rat, rabbit, and rhesus and cynomolgus monkey. 2. Seven metabolites isolated previously from man, rat, and rhesus monkey urine by preparative h.p.l.c. and identified by mass spectrometry and n.m.r. analysis were used for metabolite identification. 3. In plasma, 3-5 metabolites were found in addition to the unchanged drug. In urine, which was the main excretion route for rolipram metabolites, > 10 metabolites were detected but not the parent compound. 4. Between 40 and 70% of the compounds eliminated renally, were identified by reference to isolated metabolites. 5. Biotransformation proceeded by ether cleavage at the methoxy and pentyloxy groups, and by hydroxylation in positions 2 or 3 of the pentyloxy ring followed by sulphation. 6. In man, not in the other species studied, the 5-position of the pyrrolidone ring was also hydroxylated. This compound cross-reacted with the antibody raised against rolipram which was used formerly for the determination of rolipram in biological fluids.