To examine the responses of the levels of B₆-vitamers in several tissues to the dietary level of pyridoxine (PN), mice were fed diets containing 0, 1, 7 (the recommended level) or 35 mg PN HCl/kg diet for 5 wk. Compared with the 0 mg PN HCl/kg diet, the 35 mg PN HCl/kg diet caused the highest elevation in the concentration of pyridoxal 5'-phosphate (PLP) in small intestine and epididymal adipose tissue, moderate elevation in colon, lung, spleen and stomach, slight elevation in brain, kidney and liver (p<0.05), and no elevation in heart and gastrocnemius muscle. In general, the alterations in PLP level in many tissues and serum were remarkable for diets between 1 mg and 7 mg PN HCl/kg diets. Compared to the 7 mg PN HCl/kg diet, the 35 mg PN HCl/kg diet further elevated the PLP level in adipose tissue, spleen and stomach (p<0.05). Dietary supplemental PN elevated the level of PN in small intestine and colon in a dose-dependent manner (p<0.05), but not in other tissues. There was a significant correlation between the PN and PLP levels in small intestine and colon (p<0.05), implying that PN absorbed from the diet can be at least in part metabolized to PLP within the absorptive intestinal cells. The results suggest that the responses of concentrations of B₆-vitamers to dietary level of PN greatly differ among several tissues.