Rat everted jejunal sacs were incubated at 37 degree C in Krebs-Henseleit buffer, pH 7.4, containing thiazole-2- 14C-thiamine (0.2 micrometer), in the absence or in the presence of inorganic phosphate at a final concentration of 6mM. Control experiments under the same conditions, but without labeled thiamin were also carried out. In the intestinal sac wall, the content of labeled and endogenous (unlabeled) thiamin in the free and phosphorylated form was measured, while in the serosal fluid, total transported thiamin (labeled and endogenous) was evaluated. Accumulation in the enterocyte of inorganic phosphate, an inhibitor of thiamin pyrophosphate enzymatic hydrolysis, lowered the dephosphorylation of endogenous thiamin phosphates, both in the presence and in the absence of labeled thiamin. In the presence of labeled thiamin, the following results were observed: a) decreased labeled thiamin uptake and accumulation, both in the phosphorylated and free form; b) reduction of labeled thiamin transport to the serosal side; c) increase of endogenous thiamin exit from the intestinal cell. These findings seem to point out an important role of thiamin phosphorylation-dephosphorylation coupling in thiamin intestinal transport in vitro.