The atypical antipsychotic olanzapine was compared to other atypical as well as typical antipsychotic agents for in vivo occupancy of D1, D2, D3, 5HT2, and muscarinic receptors in rat brain. Blockade of D2 receptors was determined by measuring the levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). To assess the interaction with phosphoinositide (PI)-coupled 5HT2A and muscarinic receptors in vivo, we used a novel radiometric technique to measure in vivo PI hydrolysis. The antagonism of olanzapine and other antipsychotic agents on 5HT2A and muscarinic receptors was determined by in vivo blockade of PI hydrolysis, stimulated by the 5HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) or the muscarinic agonist pilocarpine. Olanzapine inhibited 5HT2, D2, and D3 in vivo binding with high potency (ID50=0.15, 0.6 and 1.2 mg/kg, IP, respectively), while inhibiting D1 and muscarinic in vivo binding with much less potency (ID50 > 10 mg/kg, IP). The binding of olanzapine to D2 receptors in neostriatum was well correlated with the increase of DOPAC (ED200 = 0.8 mg/kg, IP) in vivo, indicating dopamine D2 antagonism. In vivo PI hydrolysis was increased by DOI in frontal cortex and by pilocarpine in hippocampus up to 2- and 7-fold above the basal level, respectively. The agonist-induced increases in PI hydrolysis were fully blocked by the 5HT2A antagonist MDL100907 and the muscarinic antagonist scopolamine, indicating the mediation by 5HT2A receptors in frontal cortex and PI-coupled muscarinic receptors (ml, m3, and m5) in hippocampus, respectively. Olanzapine was about 8-fold more potent in vivo in blocking DOI-induced stimulation of PI hydrolysis (ID50 = 0.1 mg/kg, IP) than pilocarpine-induced stimulation of PI hydrolysis (ID50 = 0.8 mg/kg, IP). In conclusion, olanzapine is more potent in blocking the 5HT2A receptor than D1, D2, D3 and muscarinic receptors in vivo, consistent with its favorable clinical profiles. In addition, the novel in vivo PI hydrolysis assay proved to be a useful and reliable in vivo method to assess the functional efficacy of compounds that interact with the 5HT2 and muscarinic receptors.