Antisera of high sensitivity and selectivity were obtained from rabbits immunized with conjugates of hemisuccinylated fluphenazine and porcine thyroglobulin. The antiserum selected (titer 1:6000) for the development of the RIA was obtained after a priming dose and a single iv booster injection three months later. This antiserum had negligible crossreactivity with known available metabolites of fluphenazine (FPZ) and an affinity constant of 2 X 10(10) L/mol. Tritiated FPZ was further purified by HPLC and used as a ligand. The method detects as little as 20 pg/mL of plasma (4 pg/RIA tube) after 1 mL of plasma is extracted. The extraction was performed at a basic pH with heptane: isoamyl alcohol (99:1); the solvent was then back extracted using an acetic phosphate buffer. Recoveries were uniformly high (88.6 +/- 2.1%), and this aqueous buffer extract was used directly in the RIA procedure. The assay has intra- and interassay coefficients of variation of 5.8 and 8.2%, respectively, in a plasma concentration of 95 pg/mL. Results using this procedure have been cross validated against an HPLC procedure (r = 0.952, slope = 1.032, intercept = 0.009, n = 18). In a single-dose FPZ study (10 mg, po), plasma FPZ levels in 25 normal volunteers could be monitored greater than 48 h post dose. Single plasma level profiles, after an initial injection of 12.5 mg of FPZ decanoate, could be measured greater than 36 d, and, in some cases, up to 100 d post dose.