The kinetic and energetic aspects of the inhibition of taurocholate uptake by the Na+-dependent amino acid L-alanine were studied in rat basolateral liver plasma membrane vesicles. In the presence of an inwardly directed Na+ gradient, alanine (5 mM) reduced the initial velocity of taurocholate uptake to 60% of control and virtually abolished the overshoot. In the presence of a K+ gradient, the slow rate of Na+-independent taurocholate uptake was similar in the presence or absence of the amino acid. Inhibition of Na+-dependent taurocholate uptake increased nonlinearly with alanine concentration (half-maximal inhibition at approximately 1 mM) and plateaued at 5-10 mM. Kinetic studies showed that alanine significantly reduced the Vmax for taurocholate uptake from 6.32 +/- 0.22 to 3.68 +/- 0.21 nmol X mg prot-1 X min-1 but did not significantly affect taurocholate Km (38.4 +/- 3.6 vs. 29.0 +/- 4.9 microM). In contrast, the Na+-independent amino acid 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid did not affect either the initial velocity or peak uptake of taurocholate. The effects of alanine on the driving forces for bile acid uptake were directly assessed by measuring vesicle uptake of 22Na. At early time points, 22Na uptake was faster in the presence of alanine than under control conditions. These findings provide further evidence that Na+-dependent amino acids noncompetitively inhibit Na+-dependent bile acid uptake in association with accelerated dissipation of the transmembrane Na+ gradient and extend previous observations of this phenomenon made in isolated rat hepatocytes [Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G399-G403, 1983].