An enzyme immunoassay (EIA) has been developed for determination of 6-amino-5-chloro-1-isopropyl-2-(4-methyl-1-piperazinyl) benzimidazole (KB-6806), a novel 5-HT3 receptor antagonist. Anti-KB-6806 antiserum was elicited against the KB-6806-bovine serum albumin (BSA) conjugate prepared by a diazo coupling reaction through the inherent 6-amino group. Beta-galactosidase-labeled 6-amino-5-chloro-1-isobutyl-2-(4-methyl-1-piperazinyl)benzimidazole was similarly prepared by diazo coupling reaction as an enzyme-labeled antigen with a hapten heterologous combination of antiserum. The modification at the 4-methyl group of the piperazine moiety of KB-6806 significantly decreased the binding affinity to the antibody. This method could quantitate KB-6806 in dog plasma in the concentration range of 0.078-10 ng/ml with good accuracy and precision. The EIA method has been successfully applied to the determination of KB-6806 in plasma after intravenous administration of KB-6806 to dogs.