The characteristics of normal thiamine transport across the intestine were studied in rats using intact intestinal loops and everted jejunal segments. In vivo studies with [35-S]-thiamine hydrochloride revealed, in all segments of small intestine, saturation kinetics for low thiamine concentrations (0.06 to 1.5 muM), but a linear relationship between high concentrations (2 to 560 muM) and absorption. Moreover, in vitro studies of net transmural flux using everted jejunal sacs demonstrated movement of [14-C]-thiamine hydrochloride against a concentration gradient only when low, but not when high, thiamine concentration was used, so that the serosal to mucosal ratio became significantly greater than the initial value of one. Pyrithiamine, 2 muM, dinitrophenol, 200 muM, norethylmaleimide, 100 muM, and ouabain, 10 muM, reduced the net transmural flux of 0.2 muM thiamine. In contrast, these inhibitors had no effect on 20 muM thiamine. When unidirectional flux across the jejunum was measured, saturation kinetics was again demonstrated for low thiamine concentrations. This phenomenon, however, was abolished by the addition of pyrithiamine, which exerted competitive inhibition on thiamine absorption. Anoxia and sodium lack reduced intestinal uptake of 0.5 muM thiamine to 58% and 74% of normal, respectively, but did not affect uptake of 50 muM thiamine. Lowering the marked with low thiamine concentrations (O10, 1.648) than with high concentration (Q10, 1.127). Stirring of the water layer reduced Km to 59% of unstirred value, while Vmax and permeability coefficient remained unchanged. Finally, movement of low concentration thiamine against an electrical gradient was observed under conditions of electrical short circuiting and zero potential difference. In contrast, no such effect was seen with high concentrations. These studies suggest that there exists in the rat a dual system of intestinal thiamine transport. At low concentrations, thiamine is absorbed by an active process; at high concentrations, transport across the intestine is largely a passive movement.