The kinetics of mucosal membrane transport of pyridoxine hydrochloride were evaluated in vitro in the rat jejunum. Utilizing everted sacs and a double-label isotope technique, short-term incubation within the period of initial linear tissue uptake indicated: 1) no evidence of saturation of uptake over a wide pyridoxine-HCl concentration range (0.01 muM-10 mM); 2) failure of 4-deoxypyridoxine (10 muM), anoxia, iodoacetamide (5 mM), Na+ replacement and ouabain (1 mM) to inhibit uptake of 2 muM pyridoxine-HCl significantly; and 3) a low Q10 value of 1.31. Using single-label techniques, sacs were also incubated for 1 hour in 2 muM pyridoxine HCl with determination of the apparent tissue water-mucosal fluid concentration ratio and chromatographic separation of the various forms of vitamin B6 in tissue. Results demonstrated a failure of pyridoxine in tissue water to achieve a concentration in excess of that in the incubation medium. Data, therefore, were most consistent with passive diffusion as the mechanism for in vitro jejunal mucosal uptake of pyridoxine-HCl in the rat.