Betaine is accumulated in proximal renal tubular cells as an organic osmolyte. In theory, concentrative cellular uptake of betaine can be accomplished by the Na⁺,Cl^–,betaine,GABA-cotransport system (BGT-1) cloned from MDCK cells. The carrier mediates the Na⁺-coupled electrogenic transport of organic osmolytes. Cl^– may be transported together with Na⁺ and organic substrate but it is not an obligatory substrate. The expression of BGT is upregulated by osmotic cell shrinkage. In the present study, isolated perfused straight proximal tubules of the mouse were studied using electrophysiological techniques to elucidate the effects of betaine and γ-aminobutyric acid (GABA) on the potential difference across the basolateral cell membrane (PD_bl). Betaine and to a lesser extent GABA added to the bath led to the rapid depolarization of PD_bl. The betaine-induced depolarization was virtually abolished in the absence of extracellular Na⁺ and blunted in the absence of extracellular Cl^–. Inhibition of K⁺ channels by Ba²⁺ did not significantly modify betaine-induced depolarization. Increases of extracellular osmolarity enhanced, and decreases of extracellular osmolarity blunted, the betaine-induced depolarization. In conclusion, betaine transport across the basolateral cell membrane of straight proximal tubules from the mouse kidney is similar to the transport by BGT in terms of Na⁺ and Cl^– sensitivity but not the apparent affinity for GABA. The sensitivity to ambient osmolarity indicates that betaine transport in proximal tubules is regulated rapidly and nongenomically.