When sheets of mucosa from the cecum of clinically normal horses were incubated in vitro with radiolabeled L-alanine, they could accumulate this amino acid against an apparent concentration gradient after 60 to 150 minutes of incubation. The active transport system for L-alanine was on the serosal surface of the mucosal sheet only. L-Alanine accumulation at 60 minutes was partly inhibited by 20 mM glycine (P less than 0.01), 0.5 mM ouabain (P less than 0.05), and Na deprivation (P less than 0.02). Anoxia for 60 minutes increased L-alanine accumulation, but had adverse effects on cell structure and intracellular cation distributions. Transmucosal fluxes induced a small, but significant (P less than 0.05), net secretion of L-alanine, and the mean (+/- SEM) transmucosal potential difference was 7.3 +/- 0.7 mV over the period of flux measurement. It was concluded that L-alanine was accumulated by the serosal surface of the cecal mucosa, possibly to provide substrate for tissue metabolism. There was no evidence that the cecal mucosa could actively transport this amino acid from the luminal bathing medium.