It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH]m) may regulate cellular respiration rate and energetic state, it is not clear how [NADH]m itself is controlled during increased work in vivo. We have varied work and [Ca2+] in intact cardiac muscle while assessing [NADH]m using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca2+]c (by increasing [Ca2+]c or pacing frequency), [NADH]m initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH]m overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca2+]o (by increasing sarcomere length), [NADH]m fell similarly, but no recovery or overshoot was observed. This Ca(2+)-dependent recovery and overshoot may be attributed to Ca(2+)-dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH]m (since [NADH]m rapidly fell) or by [Ca2+]o (since work could also be increased at constant [Ca2+]c). However, during sustained high work, a Ca(2+)-dependent mechanism causes slow recovery of [NADH]m toward control values. This demonstrates a Ca(2+)-dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.