Antibodies were produced in rabbits immunized with physostigmine conjugated to bovine serum albumin (BSA) by two different methods: a diazo immunogen coupled to BSA with carbodiimide and a Mannich coupled immunogen. Both immunogens produced antibodies that could be used to develop sensitive and specific radioimmunoassays (RIA) for physostigmine. The limit of detection of the RIA is 100 pg of physostigmine in plasma or tissue homogenates (1.0 ng ml-1), without the need for an extraction procedure. The interassay and intraassay coefficients of variation were less than 13% in the RIA. No appreciable binding was observed between the antibodies and physostigmine metabolites or other drugs commonly used with physostigmine. The specificity of the RIA was further validated by measuring physostigmine concentrations in biological samples in tandem with high-pressure liquid chromatography. The RIA was used to study the time course of plasma concentrations and tissue distribution of physostigmine in rats.