Although recent studies have shown that combined treatment with verapamil and ATP-MgCl2 (ATP) prevents cyclosporine (CyA)-induced nephrotoxicity, the mechanism of these effects remains unknown. To study this, rat kidneys were perfused at 100 mmHg for 100 minutes with Krebs buffer containing 7.5 g/dL of albumin and substrates. After an equilibration period of 30 minutes, 500 ng/mL CyA was added. In some experiments 1 μg/mL verapamil was added 10 minutes prior to CyA and in others 2 mM ATP was added to CyA. At the end of the perfusion, cortical mitochondria (mito) were isolated and mito Ca2+ and Mg2+ (μmoles/g protein) and respiratory control ratios (RCR) were measured. In addition, total tissue Ca2+ and Mg2+ levels were measured. The results indicate that CyA treatment leads to an accumulation of mito Ca2+ and a decrease in ADP/O ratio. Simultaneous administration of ATP with CyA led to an increased mito Ca2+ accumulation and depressed RCR, which were corrected by verapamil pretreatment. The combination of verapamil pretreatment and ATP cotreatment with CyA increased tissue ATP levels from 0.8 ± 0.4 (control) to 1.4 ± 0.1 μmol/g. This pharmacologic regimen may prevent CyA-induced nephrotoxicity by preventing mito Ca2+ accumulation and by preserving mitochondrial respiratory function. This allows a more efficient generation of ATP and consequently preservation of renal function.