To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10μM) increased R340/380 by 1.1⁑0.15 (mean⁑S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19⁑0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1μM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4⁑0.06 (n=31). When 1μM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p＜0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2μM CCCP by 320⁑93.7%, compared to the control (mean⁑S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.