Rats were fed 20 or 70% casein diets with varying amounts of vitamin B6 (B6), and the B6 content and B6-dependent enzymatic activity in their tissues were examined to determine the minimum requirement of B6 for animals subjected to different levels of dietary protein (i.e., 20%: 0, 1.45, 2.90, 5.80 mg pyridoxine (PN)/kg diet; 70%: 0, 2.90, 5.80, 8.70 mg PN/kg diet). B6 requirements for the rats were almost met in the 1.45 mg PN/kg 20% casein diet and the 2.90 mg PN/kg 70% casein diet when judged from the hepatic B6 content. However, almost twice the PN was required in both 20 and 70% casein diets when judged from PLP-enzymatic activity. The content of B6 vitamers in plasma appeared to be most sensitive to B6 status, though the satisfactory level is not known. It was confirmed that, in any case tested, a high-protein diet increased the requirement of B6.