In vitro data from our laboratory demonstrate that vitamin B6 (B6) supplementation of estrogen receptor - positive and - negative breast cancer cells is growth inhibitory. Others have reported that dietary B6 supplementation resulted in increased fibrosarcoma pyridoxal phosphate (PLP) concentrations and a significant inverse relationship between tumor PLP concentration and tumor volume in mice. This suggests that, in contrast to data reported for normal cells, tumor cells are capable of accumulating supplemental B6. In the current study, we investigated the effects of dietary B6 supplementation on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats. Specifically, we aimed to identify the effect of pyridoxine (PN) supplementation on tumor growth and vitamin uptake by tumor cells. To accomplish this, 50 d old female Sprague Dawley rats were gavaged with 15 mg DMBA and fed a diet containing either 7, 350, or 1050 mg PN-HCl/kg diet, which is the equivalent of 1, 50, or 150x the National Research Council's B6 requirement for rats, respectively. These levels of PN have previously been shown to produce no overt signs of toxicity in rats. Throughout the experiment, the percent of rats with tumors and the average number of tumors per rat remained similar between groups. Mammary tumor growth rates were significantly increased in response to dietary B6 supplementation (P < 0.05). Liver PLP and pyridoxal (PL) concentrations did not differ between dietary treatment groups. Plasma PL and PLP concentrations were significantly higher in the group fed the 150x diet compared with the 1x diet (P < 0.001, P < 0.05). Mammary tissue PL concentrations of the 150x group were significantly higher (P < 0.05) than the 1x group, but no differences were observed in mammary PLP concentrations. Similarly to mammary tissue, no differences between groups were observed in tumor PLP concentration. However, tumor PL concentrations in both the 50x and 150x dietary treatment groups were significantly higher than those from the rats fed the 1x diet (P < 0.002). These data demonstrate that previously reported inhibitory effects of supplemental B6 on breast cancer growth in vitro do not occur in response to dietary supplementation at 50 or 150 times the B6 requirement in vivo. In fact, dietary B6 at 150x the requirement may actually promote mammary tumor growth. In light of these results, investigation of the effects of supplemental B6 on cancer growth in humans is warranted. Supported by American Cancer Society Grant # IRG-99-225-01.