1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.