The stereospecific binding sites of [3H]spiroperidol in frontal cortical regions of the rat brain have a higher affinity for serotonin than they do for dopamine, although the reverse relative affinity is observed in the caudate nucleus and in mesolimbic regions. The antipsychotic butyrophenones haloperidol and butaclamol compete with comparably high affinities for [3H]spiroperidol binding sites in all of the above brain regions. Both butyrophenones accelerated dopamine turnover in these three brain regions without altering serotonin turnover. A chronic treatment regimen with fluphenazine which induced tolerance to the metabolic effects of haloperidol in all three brain regions also induced tolerance to its behavioral effects. The number of binding sites of [3H]spiroperidol were increased in the caudate nucleus and mesolimbic regions but not in the frontal cortex of tolerant animals. These results are consistent with the hypothesis that [3H]spiroperidol interacts with a serotonergic site in the frontal cortex. However, the in vitro interaction of antipsychotic drugs with this receptor does not seem to be related to their acute or chronic effects on neurotransmitter function in the frontal cortex in vivo.