Immunization of New Zealand white rabbits with a bovine serum albumin conjugate of 7-hydroxy-N-carboxyethyl-N-deshydroxyethylfluphenazine produced highly specific antisera for 7-hydroxyfluphenazine (7-OHFLU). A radioimmunoassay (RIA) was developed using antisera from one of the rabbits that enabled for the first time the determination of plasma levels of 7-OHFLU, an active metabolite of fluphenazine (FLU), in patients treated with oral FLU dihydrochloride or i.m. FLU decanoate. The assay method provided sufficient sensitivity to determine accurately 20 pg of 7-OHFLU in 200 μl (0.1 ng/ml) of plasma with a coefficient of variation of <10%. The antiserum used in the RIA for 7-OHFLU demonstrated negligible cross-reactivity with FLU and its metabolites such as FLU sulfoxide, N-deshydroxyethylFLU, FLU N4'-oxide, N-deshydroxyethyl-7-OHFLU, and 7-O-glucuronide of FLU and also with other antipsychotic agents and commonly coadministered drugs. The 7-OHFLU was present in measurable amounts in all plasma samples obtained at 4-week intervals from patients receiving a daily oral dose of 5 (n = 10), 10 (n = 13), or 20 (n = 14) mg of FLU dihydrochloride. Large interindividual variations in the plasma level of FLU and 7-OHFLU were noted and the mean plasma levels ratios of 7-OHFLU/ FLU at these doses were 2.07 ± 1.08, 2.07 ± 1.13, and 2.02 ± 0.82, respectively. In the case of plasma samples from patients treated with a biweekly i.m. dose of 5 mg of FLU decanoate, 7-OHFLU could be determined in 64% of the plasma samples where the mean plasma level ratio of 7-OHFLU/FLU was 0.60 ± 0.45. These data indicate that 7-OHFLU is a major metabolite when FLU is administered either orally or intramuscularly to schizophrenic patients.