We evaluated the erythrocytes of two patients with hereditary pyrimidine 5′-nucleotidase deficiency. Significant findings included an increased reduced glutathione content, increased incubated Heinz body formation, a positive ascorbate cyanide test, and decreased intraerythrocytic pH. The pentose phosphate shunt activity of the patients' red cells as measured by the release of 14CO2 from 14C-1- glucose was decreased compared to high reticulocyte controls. Glucose-6- phosphate dehydrogenase (G6PD) activity in hemolysates from control erythrocytes was inhibited 43% by 5.5 mM cytidine 5′-triphosphate (CTP) and 50% by 5.5 mM in uridine 5′-triphosphate (UTP) at pH 7.1. CTP was a competitive inhibitor for G6P (Ki = 1.7 mM) and a noncompetitive inhibitor for NADP+ (Ki = 7.8 mM). Glutathione peroxidase, glutathione reductase, and 6-phosphogluconate dehydrogenase were not affected by these compounds. Pentose phosphate shunt activity in control red cell hemolysate at pH 7.1 was inhibited to a similar degree by 5.5 mM CTP or UTP. Since the intracellular concentrations of G6P and NADP+ are below their KmS for G6PD, these data suggest that high concentrations of pyrimidine 5′-nucleotides depress pentose phosphate shunt activity in pyrimidin 5′-nucleotidase deficiency. Thus, this impairment of the pentose phosphate pathway appears to contribute to the pathogenesis of hemolysis in pyrimidine 5′-nucleotidase deficiency hemolytic anemia.