Previous postmortem and neuroimaging studies have repeatedly suggested alterations in serotonin 5-HT(2A) receptor (5-HT(2A)R) binding associated with the pathophysiology of schizophrenia. These studies were performed with ligands, such as ketanserin, altanserin and LSD, that may bind with high-affinity to different structural or functional conformations of the 5-HT(2A)R. Interpretation of results may also be confounded by chronic antipsychotic treatment and suicidal behavior in the schizophrenia group. We quantified 5-HT(2A)R density by radioligand binding assays in postmortem prefrontal cortex of antipsychotic-free (n=29) and antipsychotic-treated (n=16) schizophrenics, suicide victims with other psychiatric diagnoses (n=13), and individually matched controls. [³H]Ketanserin binding, and its displacement by altanserin or the LSD-like agonist DOI, was assayed. Results indicate that the number of [³H]ketanserin binding sites to the 5-HT(2A)R was increased in antipsychotic-free (128 ± 11%), but not in antipsychotic-treated (92 ± 12%), schizophrenic subjects. In suicide victims, [³H]ketanserin binding did not differ as compared to controls. Aging correlated negatively with [³H]ketanserin binding in schizophrenia, suicide victims and controls. The fraction of high-affinity sites of DOI displacing [³H]ketanserin binding to the 5-HT(2A)R was increased in antipsychotic-free schizophrenic subjects. Functional uncoupling of heterotrimeric G proteins led to increased fraction of high-affinity sites of altanserin displacing [³H]ketanserin binding to the 5-HT(2A)R in schizophrenic subjects, but not in controls. Together, these results suggest that the active conformation of the 5-HT(2A)R is up-regulated in prefrontal cortex of antipsychotic-free schizophrenic subjects, and may provide a pharmacological explanation for discordant findings previously obtained.