The present studies utilized the in vivo single-pass, luminally perfused intestinal segment model in rats to evaluate pyridoxine (PN) uptake under conditions that permitted prolonged exposure of mucosa to relatively constant PN concentrations. Perfusates contained [14C]PN, unlabeled PN, and [3H]polyethylene glycol in buffer. Uptake was constant for 1 h and correlated with water absorption. Uptake of 0.2 microM PN was highest in the duodenum, intermediate in the jejunum, and lowest in the ileum. When expressed as uptake/micromolar PN, uptake of 1 mM compared to 0.2 microM PN was decreased by 37.5% in duodenum (P less than 0.001) and 14.4% in jejunum (P less than 0.05); uptake/micromolar PN were similar in ileum. In duodenum saturable uptake was apparent for 2-100 microM PN and was not explainable by a membrane carrier, presence of bile or lumen-plasma concentration gradients. Conclusions were: 1) uptake of PN by in vivo perfused intestinal segments decreases from proximal to distal; 2) the greater uptake proximally was associated with a saturable component of uptake that was greatest in duodenum, less, but significant, in proximal jejunum and absent in distal ileum and 3) saturation of uptake in the rat proximal small intestine was compatible with a role for mucosal metabolism of absorbed PN in the enhancement of uptake of PN at low luminal concentrations.