The differences of the metabolism related to vitamin B6-dependent enzymes were investigated using germ-free and conventional rats. There was a significant difference in the boyd weight gain between vitamin B6-deficient germ-free and conventional rats after about 30 days of the experiment, and the body weight gain was much less in the deficient germ-free rats than in the deficient conventional ones. Urinary excretion of xanthurenic acid was higher in the deficient germ-free rats than in the deficient conventional ones after 18 days. There was a significant difference in the activities of kynurenine aminotransferase in mitochondrial fractions of germ-free rats, but not in mitochondrial fractions of conventional ones. The activities of aspartate and alanine aminotransferases, with or without pyridoxal phosphate, significantly decreased in the deficient germ-free rats, but not in the deficient conventional ones. These findings indicate that the degree of vitamin B6 deficiency was more severe in the deficient germ-free rats than in the deficient conventional ones, and also suggest that intestinal microflora may have some effects on vitamin B6-deficient conventional rats.