Dipeptide transport was studied in chicken enterocytes and its properties compared with those of Na+-dependent sugar transport. Results showed that 1) isolated cells were capable of accumulating glycylsarcosine (Gly-Sar) against a concentration gradient (2.5- to 3.0-fold accumulation). This uptake was maximal at pH 6.0, and it was inhibited by Na+-free medium and by ouabain; 2) uptake of Gly-Sar was not affected by methionine and was competitively inhibited by carnosine, with a Ki of 12 mM; 3) the protonophore FCCP inhibited both Gly-Sar and 3-oxy-methyl-D-glucose (3-OMG) uptake by the cells; 4) amiloride, a well-known inhibitor of the Na+/H+ exchanger system stimulated 3-OMG uptake and inhibited Gly-Sar uptake, its effects being greater at pH 7.4; 5) and monensin prevents the effects of amiloride on both sugar and dipeptide uptake. In summary, Gly-Sar uptake depends on extracellular Na+ in an indirect manner via its effect on H+ efflux, and it appears to be dependent on an inward H+ gradient.