Reperfusing hearts after an ischemic episode can result in cellular Ca2+ overload. This is accompanied by the formation of contraction bands, loss of sarcolemmal integrity, and mitochondrial disruption. The present study investigated the effect of uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP) during reperfusion after 30 or 60 minutes of ischemia on this reperfusion-induced Ca2+ gain. After 60 minutes' ischemia, reperfusion with 1 mM DNP delayed the accumulation of Ca2+ and increased the duration of reperfusion before sarcolemmal disruptions were evident. This suggested that once sarcolemmal integrity is lost, Ca2+ will freely enter the cells irrespective of whether the mitochondria are able to accumulate Ca2+. After 30 minutes ischemia, reperfusion for up to 30 minutes with 0.1 or 1 mM DNP attenuated the Ca2+ gain and maintained sarcolemmal integrity. Because the authors previously found that maintaining sarcolemmal integrity alone does not totally abolish Ca2+ gain, it is suggested that DNP must prevent the entry of Ca2+ that occurs via route(s) other than those created by the loss of sarcolemmal integrity.