A sensitive and specific radioimmunoassay for normeperidine has been developed that can detect as little as 100 pg of this metabolite. In competitive binding experiments with [125I]O-tyramyl-normeperidinic acid and an antiserum produced in rabbits immunized with a bovine serum albumin-normeperidinic acid conjugate, meperidine is only 0.01% as effective an inhibitor as normeperidine. Therefore, normeperidine can be determined in physiological fluids and in tissue extracts when relatively large amounts of meperidine or other N-phenylpiperidine esters are present. High pressure liquid chromatography can be used to confirm the results obtained by radioimmunoassay during the in vitro N-dealkylations of meperidine and anileridine to normeperidine. The normeperidine isolated by high pressure liquid chromatography accounts for all of the inhibitory reactivity determined in the enzymatic digests by the radioimmunoassay. Kinetic parameters (Km and Vmax) can be determined for the N-dealkylation of meperidine and anileridine. The formation of normeperidine with time can be followed in rabbits injected with meperidine or anileridine. Plasma levels may also be determined when meperidine is administered as an obsteric analgesic.