To characterize cytosolic Ca2⁢ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200μM 2,4-dinitrophenol (DNP), and mitochondrial Ca2⁢, mitochondrial membrane potential (ΔΨm), and cytosolic Ca2⁢ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal Na⁢/Ca2⁢ exchange (NCX) in cytosolic Ca2⁢ efflux was studied in KB-R7943 and Na⁢-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by 70⁑10% within 70⁑10 s, and later by 400⁑200% at 850⁑46 s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) Ca2⁢ was depleted by 1μM thapsigargin plus 10μM ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic Ca2⁢ overload, while Na⁢-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca2⁢ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca2⁢ release from SR; 3) NCX plays an important role in transient cytosolic Ca2⁢ shifts under metabolic inhibition with DNP.