Erythrocyte pyruvate kinase (PK) from 5 patients with cogenital non-spherocytic hemolytic and erythrocyte PK deficiency have been studied by immunological methods and electrofucusing. L type immunologically related PK was titrated in crude hemolysate with anti human liver L type PK rabbit serum and M2 type immunologically related PK with anti human leukocyte M2 type PK serum. After partial purification, molecular specific activity of erythrocyte PK was measured by immunoinactivation and electroimmunodiffusion and anti L type PK serum. Partially purified erythrocyte PK was focused on continuous sucrose gradient with 2% ampholines covering the pH range 5--8. PK enzymatic deficiency was due two times to a lowered molecular specific activity of the PK variants, the concentration of PK antigen being in the normal range. In the 3 other cases enzyme activity and immunological reactivity were likewise lowered. In the 2 patients with the most marked erythrocyte PK deficiency about 50% of the residual activity in crude hemolysate were non inhibited by anti L type PK serum, but were inhibited by anti M2 type PK serum. In 3 patients, the electrofocusing pattern of partially purified PK was significantly different from than of normal controls. In conclusion, the heterogeneity of the molecular mechanisms of the deficiency on the one hand, and the abnormalities of electrofucusing patterns on the other hand, seem to indicate that erythrocyte PK deficiency is due to the synthesis by muted structural genes of various abnormal PK molecules.