A rapid, sensitive, precise radioimmunoassay for conjugates of chenodeoxycholic (chenic) acid has been developed and validated. Immunogen was prepared with chenylglycine couples to bovine serum albumin, emulsified in Freund's complete adjuvant, and injected intracutaneously in rabbits. Antibodies of moderate titer (used at 1:7500 final dilution) were obtained. Tracer of high specific activity was prepared by reductive tritiation of delta11-chenylglycine. The binding step required 1 hr at room temperature; separation of bound tracer was achieved by addition of ammonium sulfate. The limit of sensitivity of the assay was about 20 nmoles per liter, and the lower limit of the working range was 0.4 mumole per liter. The assay was validated by measuring levels in samples of fasting state serum to which known amounts of chenylglycine had been added; the assay was also validated by means of gas-liquid chromatography on sera from jaundiced patients. The median fasting state serum level in 56 Caucasians and blacks was 0.9 mumole per liter (upper limit of normal (95% confidence) being 2.3), which is in the same range as that reported by others using gas-liquid chromatography. A 5- to 10-fold increase in levels of chenyl conjugates was observed 1 hr after the ingestion of a liquid meal in healthy persons.