The interaction of the psychotropic agent olanzapine with serotonin 5-HT 3 and 5-HT 6 receptors was investigated. Olanzapine did not contract the isolated guinea pig ileum, but blocked contractions induced by the 5-HT 3 receptor agonist 2-methyl serotonin (2-CH 3 5-HT) with a pK B value of 6.38+/-0.03, close to the affinity of the 5-HT 3 receptor antagonist ondansetron. The atypical antipsychotic risperidone (1 μM) did not significantly inhibit 2-CH 3 5-HT-induced contractions. Olanzapine had high affinity (pK i =8.30+/-0.06) for human 5-HT 6 receptors in radioligand binding studies. Olanzapine did not stimulate [ 3 5 S]guanosine-5'-O-(3-thio)triphosphate ([ 3 5 S]GTPγS) binding to the G protein G s in cells containing human 5-HT 6 receptors, but inhibited 5-HT-stimulated [ 3 5 S]GTPγS binding (pK B =7.38+/-0.16). Among other antipsychotics investigated, clozapine antagonized 5-HT 6 receptors with a pK B =7.42+/-0.15, ziprasidone was three-fold less potent, and risperidone, quetiapine and haloperidol were weak antagonists. Thus, olanzapine was not an agonist, but was a potent antagonist at 5-HT 6 receptors and had marked antagonism at 5-HT 3 receptors.