Cross-bridges exist in a number of transient states, each differing in their biochemical and structural properties, during each cycle of ATP hydrolysis. Techniques pioneered in our laboratory allow measurement, in real time, of changes in the distribution of cross-bridge states as a function of physiological (e.g. cross-bridge strain) and physical (e.g. temperature) determinants of muscle function (1, 2). Our aim here was to use these methods to characterize the mechanoenergetic properties of rat cardiac trabeculae. Inorganic phosphate (Pi) release, and therefore actomyosin (AM) ATPase rate, was determined during contractions of Triton-permeabilized trabeculae at 20°C. Female Sprague-Dawley rats were killed by cervical dislocation and thin, unbranched, uniform trabeculae dissected from the left ventricle. Initial sarcomere length (SL) was set to 1.9µm by laser diffraction, contraction was elicited by laser-flash photolysis of NPE-caged ATP (P3-l(2-nitrophenyl)ethyladenosine-5′-triphosphate) and time-resolved Pi release monitored using the fluorescent protein MDCC-PBP, (N-(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. Isometric force produced during maximal activation (32µM Ca2+; n=5) was 50.8±6.4 kNm-2. The ATPase rate during the first AM turnover (assuming 150 µM myosin heads) was 12.1±1.0s-1, which decreased to a steady state of 6.5±0.4s-1 once the isometric plateau had been reached. This is comparable to previous steady state measurements, using an NADH-linked enzyme assay for the rate of ADP appearance, of 10s-1 (3) and 3.3s-1 (4). At submaximal activation (3µM Ca2+; n=6) isometric force was reduced to 43.2±5.2 kNm-2, initial ATPase rate was 13.2±1.5s-1 and steady state ATPase rate was 6.72±0.4s-1. During lengthening (5% of muscle length, L0; 0.5 L0s-1; SL=2.1µm) the rate of Pi release, and therefore AM ATPase rate decreased to 1.6±0.9s-1 at maximal activation (n=5) and 3.0±0.7s-1 at submaximal activation (n=6). After stretch, the ATPase rate at SL 2.1µm was 4.0±0.3s-1 at maximal activation and 3.6±0.2s-1 at submaximal activation. Shortening (5% L0; 0.5 L0s-1) caused a transient increase in Pi release rate to 7.5±1.7s-1 at maximal activation and 7.0±0.4s-1 at submaximal activation. ATPase rate declines slowly throughout the experiment due to ADP accumulation and ATP depletion, explaining only a small increase in ATPase rate during shortening compared with steady state. Our previous work has shown similar responses to lengthening and shortening of permeabilized psoas fibres. The slower Pi release during the stretch is due to the forcible detachment of cross-bridges through a reversal of the power stroke, resulting in less ATP being hydrolysed. The faster Pi release rate during shortening is brought about by acceleration of the rate of ADP release from AM at low strain, resulting in faster cross-bridge cycling.