Tubuli were prepared from rat renal cortex by modification of the method adopted from HOWARD and PESCH (J.Biol. Chem. 243, 3105, 1968). At 37 degrees C tubuli showed constant pyruvate consumption and glucose production for more than one hour. Intracellular electrolyte and amino acid concentrations were measured after separating the tubuli from the incubation medium by a layer of silicon oil in 5 seconds. During incubation at 37 degrees C L-alanine was accumulated 2.6 fold in the tubuli, while intracellular Na+ concentration dropped to about 50 mEq/l. At 27 degrees C, when pyruvate consumption and glucose production was reduced to less than half compared with 37 degrees C incubation, the respective gradients were higher. L-alanine accumulation was reduced by 2,4-dinitrophenol, antimycin A, ouabain and p-hydroxymercuribenzoate and was completely abolished by the reversal of the Na+ concentration gradient between extracellular and intracellular spaces. At 4 degrees C, when erergy production in tubuli ceased, L-alanine could be concentrated in the case of a Na+o/Na+i ratio greater than unity but not at a lower ratio. It is concluded that the accumulation of L-alanine is a direct function of the energy provided by the Na+ concentration gradient and is mediated by a postulated carrier, which can be injured by sulfhydryl group reagents like p-hydroxymercuribenzoate.