We have previously shown that increased cardiac work initially caused a rapid Ca(2+)-independent fall of mitochondrial [NADH] ([NADH](m)) to a minimum level, and this was followed by a slow Ca(2+)-dependent recovery toward control level (Brandes and Bers, Biophys. J. 71:1024-1035, 1996; Brandes and Bers, Circ. Res. 80:82-87, 1997). The purpose of this study is to improve our understanding of the factors that control [NADH](m) during increased work. [NADH](m) was monitored using fluorescence spectroscopy in intact rat trabeculae isolated from the right ventricular wall. Work was increased by increasing sarcomere length, pacing frequency, external [Ca(2+)], or by decreased temperature. The results were: 1) The initial fall of [NADH](m) during increased pacing frequency depends independently on increased myofilament work and on increased Ca(2+)-transport ATPase activity. 2) The [NADH](m) recovery process depends on average cytosolic [Ca(2+)] (Av[Ca(2+)](c)), but not on absolute work level. 3) The initial fall of [NADH](m) and the [NADH](m) recovery are similar whether increased work is associated with low frequency and high Ca(2+)-transient amplitude or vice versa (at the same myofilament work level and Av[Ca(2+)](c)). 4) The mechanisms associated with the smaller fall and recovery of [NADH](m) at 37 degrees C versus 27 degrees C, may be explained by lowered Av[Ca(2+)](c) and myofilament work. The NADH control mechanisms that operate at lower temperature are thus qualitatively similar at more physiological temperatures.