This study in well-oxygenated, freshly isolated rat proximal tubules (RPT), examined the effects of several drugs that alter the transmembrane K+and Na+gradients across cell membranes, including valinomycin (VAL), amphotericin B (AMPHO), and ouabain (OUAB). The effects of high extracellular potassium chloride (KCl) concentration (45 mM) and low extracellular sodium concentration (100 mM) were also studied. After 10 min of drug exposure Ca2+uptake rate (nmol/mg/min) increased from 2.7 to 3.8 with VAL (p<. 02), from 2.9 to 3.7 with AMPHO (p<. 05), from 3.6 to 4.1 with OUAB (p<. 05), and from 3.2 to 4.8 with 45 mM KCl (p<. 001). Ca2+uptake rate was sustained at these high levels at 20 min in all treated RPT except those exposed to OUAB. LDH release averaged less than 15% in control tubules and did not increase significantly except in RPT treated with VAL, where LDH release at 10 min was 48% and at 20 min was 57% (both p<. 001). Of importance, only in VAL-treated RPT did ATP decrease to low levels (6.7 nmol/mg in control to 2.0±0.3 nmol/mg in VAL, p<. 001). Treatment with verapamil reduced Ca2+uptake rates at 10 min in VAL-treated RPT (from 3.8 to 3.1, p<. 02, in AMPHO-treated RPT (from 3.8 to 3.1 p<. 001), in OUAB-treated tubules (from 4.0 to 3.4, p<. 01), and in KCl-treated RPT (from 3.7 to 3.2, p<. 01). These results indicate that acute changes in the transmembrane ion gradient in RPT are accompanied by increased Ca2+uptake rates. Ca2+uptake rates are also increased during O2deprivation in RPT, a situation in which the transmembrane ion gradient is likewise altered. The increased Ca2+uptake rate observed in the present study and during hypoxia may have a common basis, that is, altered transmembrane ion gradients or some function thereof.