A radioimmunoassay was developed for measuring plasma concentrations of the antihypertensive agent guanethidine at the nanogram level. Guanethidine was conjugated covalently to human serum albumin by two procedures, and the degree of conjugation was determined using tracer amounts of 3H-guanethidine. Immunization of sheep ethidine, as determined in competitive binding studies using 3H-guanethidine and a dextran-coated charcoal technique for the separation of free and antibody-bound drug. The major human metabolities, an N -oxide and a ring-opened derivative, were not cross-reactive in antibody binding studies. Constituents of human plasma or serum do not appear to interfere with the assay. Preliminary results from immunoassay of plasma samples from patients receiving guanethidine indicate potential use for assessing dosage regimens and studying pharmacokinetics.