Although ischemia-reperfusion (I/R) has been shown to depress cardiac performance and sarcoplasmic reticulum (SR) function, the mechanisms underlying these alterations are poorly understood. Because lack of oxygen and substrate deprivation are known to occur during the ischemic phase, we examined the effects of reperfusion on cardiac performance and SR function in hearts subjected to hypoxia and substrate lack. For this purpose, isolated rat hearts were perfused with hypoxic and/or glucose-free medium for 30 min and then reperfused with normal medium for 1 h; the SR vesicles were isolated for studying the Ca²⁺-transport activities. Reperfusion with normal medium of hearts deprived of oxygen or glucose showed no changes in cardiac performance and SR function. However, reperfusion of hearts perfused with hypoxic glucose-free medium showed ~45% decrease in cardiac contractile activities as well as 23 and 64% reduction in SR Ca²⁺-uptake and Ca²⁺-release activities, respectively, without any change in the level of SR Ca²⁺-cycling proteins. Depressed SR function in these hearts was associated with a reduction in Ca²⁺/calmodulin-dependent protein kinase (CaMK) phosphorylation of the SR Ca²⁺-cycling proteins and 34% decrease in SR CaMK activity. These changes in cardiac performance, SR function, and SR CaMK activity in the hypoxic, glucose-deprived, reperfused hearts were similar to those observed in hearts subjected to 30 min of global ischemia and 60 min of reperfusion. The results therefore suggest that the lack of both oxygen and substrate during the ischemic phase may contribute to the I/R-induced alterations in cardiac performance and SR function. Furthermore, these abnormalities were associated with reduced SR CaMK activity.