Pyrimidine-5''-nucleotidase deficiency appears to be an important cause of hemolytic anemia associated with basophilic stippling of the red cells. A new radiometric method for the assay of this enzyme was developed. In this technique, 14C-CMP served as substrate. The CMP which was not dephosphorylated to cytidine was bound to the barium sulfate precipitate which formed in the deproteinization process. The cytidine remained in solution and was counted. The method was simple and reproducible and could be carried out on large numbers of samples. Two patients with pyrimidine-5''-nucleotidase deficiency were detected by means of this technique.