The antidopaminergic properties of thioridazine (THD) and its major metabolites were evaluated using in vitro and in vivo estimates of dopaminergic function. THD-2-sulfone was more potent than, and THD-2-sulfoxide equipotent to, THD in displacing spiperone from rat striatal membranes and in inhibiting dopamine-stimulated cyclic adenosine 3',5'-monophosphate synthesis in rat striatal homogenates. Other major THD metabolites were relatively inactive. These in vitro data suggest that THD, THD-2-sulfone and THD-2-sulfoxide are potent dopamine receptor blocking agents. Intraperitoneally administered THD antagonized amphetamine-induced locomotion and also increased the concentration of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in terminals of the nigrostriatal dopamine system. Conversely, THD administered i.c.v. into conscious animals, did not antagonize amphetamine-induced locomotion nor increase brain regional concentrations of DOPAC or HVA. On the other hand, the i.c.v. administration of THD-2-sulfone and THD-2-sulfoxide dose-dependently inhibited amphetamine-induced locomotion and also increased the concentrations of HVA and/or DOPAC in the striatum and olfactory tubercles. These apparently discrepant in vitro and in vivo data suggest that the biotransformation of THD is a major determinant in those actions of THD attributed to a blockade of dopamine receptors in the central nervous system.