The vascularly perfused small intestine and hind limb muscle of the rat were utilized to study the transport and metabolism of pyridoxine (PN), independent of other tissues, including erythrocytes. The transport of PN both into the mucosal tissue and transmurally into the perfusate was proportional to the dose over a 10,000-fold range of concentrations. The only labeled compound formed from [3H]-PN in the mucosa was PNP which accounted for 30.6% of the isotope found there. The data for the hind limb muscle suggest that transport occurs by passive diffusion followed by phosphorylative trapping. Over a 10,000-fold range of concentrations of PN in the perfusate, the percentage of 3H found in the muscle ranged from 10.4 to 15.7 for 30-minute experiments. As the dosage was increased the percentage of 3H in the muscle, present as PN increased and that in PNP decreased. In longer experiments, up to 75 minutes, with 20 nmole of [3H]-Pn, the PN in muscle decreased as phosphorylation occurred. There was no evidence of any conversion of PNP to PLP (pyridoxal phosphate) in the perfused hind limb.