Antiserum against 3-O-methyldopamine (MD) was produced in rabbits immunized with MD hapten conjugated to hemocyanin. The antiserum was used to develop a radioimmunoassay (RIA) for MD. As little as 0.5 ng of MD in 0.1 ml can be detected. The major catecholamines and the phenolic aromatic amines (dopamine, norepinephrine, epinephrine, octopamine, and tyramine) and their metabolites (normetanephrine, metanephrine, homovanillic acid and 4-hydroxy-3-methxymandelic acid) did not bind significantly to the antibody. The RIA of MD was used to assay the endogenous level of MD in urine and plasma of hospitalized children. In children (7 mo to 13 yr), average concentration of MD in plasma was found to be 0.47 +/- 0.11 ng/ml, and in urine 0.15 +/- 0.05 microgram/mg of creatinine (45.0 +/- 16.3 microgram/24 hr). In children with neuroblastoma, there was a 3- to 10-fold increase in urinary excretion and plasma level of 3-O-methyldopamine. In adults, the average urine and plasma levels were found to be 87.4 +/- 3.4 microgram/24 hr and 0.6 +/- 0.2 ng/ml. The diagnostic applicability of the RIA of MD is discussed.