A simple and highly sensitive radioimmunoassay (RIA) procedure has been developed for the determination of the new antihypertensive drug 2-[(2-chloro-4-methyl-3-thienyl)amino]-2-imidazoline (tiamenidine) in serum, plasma and urine. Antisera to tiamenidine were produced in rabbits against its derivatives conjugated to bovine serum albumin. Studies with metabolites and imidazoline derivatives have shown that the antibodies thus produced are specific for tiamenidine. 3H-Labelled drug was used as tracer. The separation of free from antibody-bound tiamenidine was carried out by using polyethylene glycol. The radioimmunoassay (RIA) allows the determination of tiamenidine in 100 microliter biological specimens directly and without extraction down to a detection limit of 20 pg/ml. Reproducibility and accuracy of the assay are good. A sufficient correlation (r = 0.95) was obtained when serum samples were assayed by the RIA and the GC-MS method. The pharmacokinetic profile of the drug was determined in subjects after oral administration of 1 mg tiamenidine using the newly developed RIA. The course of serum levels up to 24 h after treatment may be well described by a Bateman function with an elimination half-life of about 3-5 h. These values correspond sufficiently well with the data obtained from the rate constant of the excretion via urine.