A sensitive radioimmunoassay for amitriptyline and nortriptyline in blood has been developed. The antibodies used in the radioimmunoassay were raised in a sheep against a conjugate of nortriptyline and bovine serum albumin. Using tritiated amitriptyline as the label, the assay is capable of detecting concentrations as low as 2.0 ng/ml in a 50 microliter sample of plasma. Cross-reactivity studies have demonstrated the specificity of the radioimmunoassay for both amitriptyline and nortriptyline, and comparison with gas-liquid chromatography assay has indicated the applicability of the assay to a routine situation. The radioimmunoassay has been used to study the plasma drug levels after single oral administration of amitriptyline to four volunteers. A wide variation in maximum drug concentrations, ranging from 18 to 62 ng/ml, was seen, with the time taken to reach the maxima ranging between 1.5 and 3 hours. A second concentration peak was seen in three of the volunteers, at 4 to 5 hours after ingestion of the drug.