Reperfusion injury of cardiac myocytes following metabolic inhibition (MI) is characterised by hypercontracture, loss of Ca²⁺-homeostasis and contractile function, and is sensitive to the energetic status of the heart (Rodrigo et al. 2002). We investigated the role of mitochondrial re-energization in reperfusion injury by studying cellular events during reperfusion following MI, where NADH and FADH₂ concentrations are high, and uncoupling where mitochondria are fully oxidized.Ventricular myocytes were isolated from humanely killed adult rats (Rodrigo et al. 2002) and stimulated at 1 Hz and at 35°C. Contraction was measured with a video system, [Ca²⁺]_i with fura-2 and [ATP]_i with Mg green. For MI we used substrate-free Tyrode containing 2mM NaCN + 1 mM IAA, and uncoupling used 10 mM CCCP. Both MI and uncoupling caused contractile failure, rigor and an increase in diastolic [Ca²⁺]_i. After 10 min MI [Ca²⁺]_i was 256 ± 5 nM (mean ± S.E.M. n = 25 experiments) in MI, and 489 ± 18 nM (n = 27) during uncoupling. Reperfusion of MI-treated myocytes with normal Tyrode induced an immediate but transient fall in [Ca²⁺]_i to 179 ± 6 nM (n = 14) followed by a hypercontracture. However, reperfusion after uncoupling had no immediate effect on cell length or [Ca²⁺]_i. Rather, there was a delay of 2-3 min before a transient fall in [Ca²⁺]_i accompanied by a hypercontracture. Following hypercontracture [Ca²⁺]_i climbed steadily in both MI and uncoupled cells, reaching 300-350 nM after 10 min reperfusion. Reperfusion of MI and uncoupled myocytes resulted in a transient increase in ATP, which preceded hypercontracture. However, ATP concentration continued to remain low in MI-treated myocytes but recovered to near control values in uncoupled myocytes. Removal of extracellular Ca²⁺ during MI or uncoupling limited the rise in [Ca²⁺]_i to 127 ± 12 nM (n = 11), and 100 ± 5 nM (n = 11) respectively, but had no effect on reperfusion-induced hypercontracture. Removing Ca²⁺ during reperfusion prevented the reperfusion-induced increase in [Ca²⁺]_i in both cases, but only blocked hypercontracture in uncoupled myocytes.Our results support a model for reperfusion injury of MI-treated myocytes where production of ATP secondary to mitochondrial reenergization is primarily responsible for initiating hypercontracture by activating cross-bridge cycling. The reduced severity of reperfusion injury in uncoupled myocytes may be linked to loss of mitochondrial reducing power (NADH, FADH₂)