The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl2, KH2PO4 and MgSO4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Herpes-mannitol buffer showed two entry systems for proline in each buffer with similar Km values, but the maximal rate of transport (V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.