We have investigated the effects of 2,3-butanedione monoxime (BDM) and mannitol on Ca2+ metabolism in rabbit cardiac muscle. Simultaneous measurements of force and intracellular Ca2+ were made in right ventricular papillary muscles loaded with the fluorescent Ca2+ indicator fura-2. At a BDM concentration of 2 mM, peak isometric force was only 52% of control values and this was reduced to 18% at a concentration of 5 mM. The peak of the Ca2+ transient decreased by 8% at 2 mM BDM and by 18% at the higher concentration. In the presence of 362 mM mannitol peak isometric force decreased by 78% and there was a tendency for the peak of the Ca2+ transient to increase. A combination of 362 mM mannitol with 5 mM BDM completely inhibited force production despite peak Ca2+ levels that were no different from control values. In myothermic experiments under similar conditions the latency release protocol of Gibbs et al. (1988) and the BDM protocol of Alpert et al. (1989) were used to derive independent estimates of tension-independent (activation) heat in the same muscle. For both protocols the heat-stress relationship was well fitted by first-order linear regression. The activation heat estimate was significantly higher when measured with the latency release technique (2.31 mJ/g) compared with the BDM protocol (1.24 mJ/g). Our results confirm that in rabbit cardiac muscle low concentrations of BDM (2 mM) cause a marked inhibition of force development with little apparent effect on peak Ca2+ levels. Therefore, the lower activation heat estimates under these conditions may not be due to a reduced intracellular Ca2+ concentration. It is possible that the higher activation heat values obtained with protocols not involving chemical interventions may include the energy usage contributed by cellular processes that presumably do not occur in the presence of BDM and mannitol.