We studied sodium-dependent uptake of L-alanine into small intestinal brush border membrane vesicles (BBMV) isolated from piglets 40 h after infection with transmissible gastroenteritis (TGE) virus. Vesicles from TGE-infected pigs and uninfected litter-mate controls showed comparable degrees of enrichment and purity. In BBMV prepared by conventional techniques, [3H]L-alanine "overshoot" (peak uptake/equilibrium uptake) in the presence of a Na gradient was preserved in TGE BBMV, unlike [3H]D-glucose "overshoot," which was reduced. When these experiments were repeated using vesicles of greater purity, initial rates of Na-dependent L-alanine influx were reduced in BBMV from infected piglets under voltage clamped conditions with valinomycin. These studies demonstrate a specific amino acid transport defect in the small intestinal epithelium during acute viral diarrhea. They demonstrate too that brush border L-alanine-Na co-transport, although reduced, is present after viral damage, confirming previous studies that showed additive effects of amino acid and glucose on jejunal epithelial Na+ transport in transmissible gastroenteritis. Our findings support the concept that, in viral enteritis, oral rehydration solutions containing amino acid and glucose have a theoretical advantage over glucose electrolyte solutions because they facilitate brush border Na+ entry by two carrier mechanisms.