The active Na⁺-independent transport of L-alanine across the duodenal mucosa of the lizard Gallotia galloti was studied in Ussing-type chambers using a computer-controlled voltage clamp. Addition of L-alanine to the Na⁺-free bathing solutions resulted in a significant L-alanine absorption (J_net) that was paralleled by an increase in transepithelial short-circuit current (I_sc) and potential difference (PD) without apparent changes in the tissue conductance. The concentration dependence of J_net, PD, and I_sc displayed Michaelis-Menten kinetics. L-alanine-induced electrical changes were completely inhibited by external alkaline pH or by the H⁺-ionophore carbonyl cyanide m-chlorophenyl-hydrazone in the bathing solution. The alanine-induced electrogenicity was dependent on the presence of extracellular K⁺ and could be blocked by serosal Ba²⁺ or mucosal orthovanadate. These results suggest the existence of an H⁺-coupled L-alanine cotransport at the apical membrane of enterocytes. The favorable H⁺ driving force is likely to be maintained by an apical vanadate-sensitive H⁺-K⁺-ATPase, allowing the extrusion of H⁺ in an exchange with K⁺. Potassium exit through a basolateral barium-sensitive conductance provides the key step for the electrogenicity of L-alanine absorption.