1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance. 2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5-500 microM). Slices gave consistently higher Km's (20 and 30 microM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 microM respectively) and microsomes (2 and 5 microM respectively.) The rank order of Vmax and CL(int) was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor. 3. The high CL(int) of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL(int) (9 ml/min/SRW).