Octoclothepin (1) was resolved into its R and S enantiomers via the diastereomeric tartaric acid salts. The enantiomers were shown to be of high optical purity by 1H NMR with use of the chiral shift reagent (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol. Pharmacological and biochemical testing confirmed that (S)-1 is the more potent dopamine (DA) D-2 antagonist both in vitro and in vivo, although the R enantiomer still has significant D-2 antagonistic activity. In contrast, both enantiomers were equally active in test models detecting activity at D-1 receptors, serotonin-2 (5-HT2) receptors and alpha 1 adrenoceptors. Contrary to a previous prediction, it was found that norepinephrine (NE) uptake inhibition was confined solely to the S enantiomer. Overall, (S)-1 has a "classical" neuroleptic profile, while the R enantiomer has a more "atypical" profile. These pharmacological profiles seem to be in agreement with the reported clinical profiles of the two enantiomers. A previous conformational study was revised in light of the biochemical test results with enantiomers of known optical purity. Their relative D-2 receptor affinity corresponded well with the calculated conformational energy difference between their "active conformations" deduced from a previously reported new D-2 receptor model. Also the high enantioselectivity of (S)-1 at the NE uptake site could be explained after a detailed conformational analysis showing strict requirements for the orientation of the piperazine lone-pair direction at the NE uptake site.