TNFα levels are increased during intestinal inflammation processes and malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose absorption in rat and rabbit intestinal everted rings and other authors have shown regulation of PET1, TAUT and SERT transporters by the cytokine in Caco-2 cells. Thus, the aim of the present work was to investigate in Caco-2 cells the effect of TNFα on the absorption of alpha-methyl-D-glucoside (MG), glutamine and phenyalanine, and the possible implication of PKA and PKC. Caco-2 cells were grown on plates (access of the cytokine from the apical membrane) or on filters (access from both apical and basal membranes). Cells were preincubated for diverse times with different concentrations of TNFα in the basal or apical medium, before measuring the uptake of the radiolabeled substrate during 15 min. In the experiments with kinases inhibitors, cells grown on plates were preincubated for 30 min with 2 μM chelerythrine (PKC inhibitor) or 1 μM H-89 (PKA inhibitor) prior to the 15 min incubation with 25 or 50 ng/ml TNFα. MG was chosen as specific substrate of the Na⁺/glucose cotransporter SGLT1, Phe as specific substrate of B⁰AT1 and Gln as substrate of both ASCT2 and B⁰AT1. The results with plates-grown cells demonstrated inhibitory effect of TNFα (with 10 ng/ml and higher concentrations) on 0.1 mM MG uptake, even without preexposure to the cytokine. After 24 h preincubation, the inhibition was observed already with 1ng/ml. When filters were used, the presence of 10 or 25 ng/ml TNFα in the basal medium during 1h preexposition increased MG uptake, whereas after 24 h, 1 ng/ml inhibited uptake and no effect was found with 10 ng/ml TNFα. The apical presence of TNFα (10, 25 or 50 ng/ml) diminished the uptake of 0.1 mM Gln and 0.1 mM Phe by Caco-2 cells grown on plates. In filter-grown cells, the presence of TNFα (10-50 ng/ml) in the basal compartment during 1, 6 or 24 h preincubation did not modify the uptake of Gln or Phe. In studies performed using plates, both H-89 and chelerythrine reversed the inhibition of MG uptake due to 25 ng/ml TNFα whereas they increased the inhibition of Gln and Phe uptake due to the cytokine. In summary, TNFα inhibits SGLT1, B⁰AT1 and probably ASCT2 function in monolayer of Caco-2 cells. The effect is stronger when the cytokine is acting from the apical membrane. Both PKA and PKC seem to be involved in the apical effect of the cytokine on SGLT1.