The atypical antipsychotic olanzapine has relatively high affinity for a number of neuronal receptors in radioreceptor binding assays. The ability of olanzapine to activate or antagonize a number of neuronal receptors was investigated in vitro, in cell lines transfected selectively with receptor subtypes and in receptor-selective isolated tissue studies. Olanzapine had no agonist activity at any of the receptors examined. However, olanzapine was a potent antagonist of 5-HT-stimulated increases in IP₃ in cell lines transfected with 5-HT_2A or 5-HT_2B receptors with IC₅₀ values of 30–40nM. Olanzapine weakly blocked 5-HT-induced formation of IP₃ in cell lines transfected with 5-HT_2C receptors, but in this cell line potently inhibited 5-HT-stimulated [³⁵S ]GTPγS binding with a K_i value of 15nM. Olanzapine blocked dopamine-stimulated adenylyl cyclase in rat retina with modest potency (K_i=69nM), consistent with its relatively low affinity for dopamine D1 receptors. Olanzapine blocked agonist-induced activities at the muscarinic receptor subtypes M₁, M₂, M₃, and M₅ with K_i values of 70, 622, 126, and 82nM, respectively. In studies using cell lines transfected with muscarinic M₄ receptors, olanzapine and the atypical antipsychotic clozapine did not have agonist activities as determined with cAMP inhibition and stimulation assays, arachidonic acid release and [³⁵S ]GTPγS binding assays. However, olanzapine antagonized agonist-induced effects in muscarinic M₄ cells with a K_i value of 350nM. In isolated tissue studies, olanzapine potently blocked agonist-induced effects at α₁-adrenergic and histamine H₁ receptors (K_B=9 and 19nM, respectively). Thus, olanzapine was an antagonist at all receptors investigated and was a particularly potent antagonist at 5-HT_2A, 5-HT_2B, 5-HT_2C, α₁-adrenergic and histamine H₁ receptors. Olanzapine was a weaker antagonist at muscarinic and dopamine D1 receptors.