Summary: A radioimmunoassay for encainide (4-methoxy-2‘-[2-(lmethyl-2-piperidyl)ethyl]benzanilide hydrochloride), a new antiarrhythmic agent, has been developed by immunization of rabbits with an O-carboxymethyl analog of encainide conjugated to bovine serum albumin. A radioactive tracer containing 125I was prepared by chloramine-T iodination of the tyramine amide of the O-carboxymethyl analog. Separation of bound from free tracer in the assay is accomplished using a second antibody. The assay is capable of detecting 20 pg of encainide directly or 0.2 ng of encainide/ml of plasma or urine. Interassay variability of the slope, y-intercept, and 50% bound points on the standard curve had coefficients of variation (CV) of 6.9, 7.8, and 6.0%, respectively. Mean recoveries of 94.4% with a CV of 12.0% were obtained after extraction and analysis of standards from plasma at concentrations of 20, 200, and 2,000 ng/ml. No interference with the assay was observed from any components in normal, hemolyzed, or turbid plasma or from urine. The extent of cross-reactivity of possible metabolites of encainide was evaluated, and the assay for unchanged encainide has been validated by high pressure liquid chromatography (HPLC). A HPLC procedure for the quantification of encainide in plasma is also described. Encainide and three putative metabolites, the O-demethyl, N-demethyl, and N,O-bis-demethyl congeners of encainide, can be quantified simultaneously by this procedure. All four compounds are extracted from buffered plasma (pH 8.5) with n-butyl chloride containing 5% isopropyl alcohol (v/v). The compounds are then separated by HPLC on a μPorasil column and quantified by measuring their uv absorbance at 254 nm. Each compound can be