Atypical antipsychotic drugs, which are more potent direct acting antagonists of brain serotonin (5-HT)(2A) than dopamine (DA) D(2) receptors, preferentially enhance DA and acetylcholine (ACh) efflux in the rat medial prefrontal cortex (mPFC) and hippocampus (HIP), compared with the nucleus accumbens (NAc). These effects may contribute to their ability, albeit limited, to improve cognitive function and negative symptoms in patients with schizophrenia. Asenapine (ASE), a new multireceptor antagonist currently in development for the treatment of schizophrenia and bipolar disorder, has complex serotonergic properties based upon relatively high affinity for multiple serotonin (5-HT) receptors, particularly 5-HT(2A) and 5-HT(2C) receptors. In the current study, the effects of ASE on DA, norepinephrine (NE), 5-HT, ACh, glutamate, and gamma-aminobutyric acid (GABA) efflux in rat mPFC, HIP, and NAc were investigated with microdialysis in awake, freely moving rats. ASE at 0.05, 0.1, and 0.5 mg/kg (s.c.), but not 0.01 mg/kg, significantly increased DA efflux in the mPFC and HIP. Only the 0.5 mg/kg dose enhanced DA efflux in the NAc. ASE, at 0.1 and 0.5 mg/kg, significantly increased ACh efflux in the mPFC, but only the 0.5 mg/kg dose of ASE increased HIP ACh efflux. ASE did not increase ACh efflux in the NAc at any of the doses tested. The effect of ASE (0.1 mg/kg) on DA and ACh efflux was blocked by pretreatment with WAY100635, a 5-HT(1A) antagonist/D(4) agonist, suggesting involvement of indirect 5-HT(1A) agonism in both the actions. ASE, at 0.1 mg/kg, increased NE, but not 5-HT, efflux in the mPFC and HIP. ASE, at 0.1 mg/kg (s.c.), had no effect on glutamate and GABA efflux in either the mPFC or NAc. These findings indicate that ASE is similar to clozapine and other atypical antipsychotic drugs in preferentially increasing the efflux of DA, NE, and ACh in the mPFC and HIP compared with the NAC, and suggests that, like these agents, it may also improve cognitive function and negative symptoms in patients with schizophrenia.