The kinetics of ATP hydrolysis and tension responses were studied simultaneously in a permeabilized preparation of cardiac tissue of the guinea pig. This was achieved by combining laserflash photolysis of P3-1-(2-nitrophenyl)ethyladenosine 5'-triphosphate ("caged-ATP") and a rapid freezing technique. In the presence of calcium ions, tension increased following the photolytic production of ATP with a half-time of 0.3 s. The timecourse of ATP hydrolysis consisted of an initial rapid phase followed by a steady-state hydrolysis rate of 0.4 s-1, indicating that the rate-limiting step of the ATPase in isometric fibers is slower and subsequent to the nucleotide hydrolysis step: the isometric steady state intermediate is probably an actomyosin-ADP complex. In the absence of calcium ions, rigor tension decreased upon the photolytic production of ATP with a half-time of 0.45 s. The time course of ATP hydrolysis was biphasic with a rapid initial phase of ATP hydrolysis, followed by a steady-state hydrolysis rate which was too slow to measure over the time scale of these experiments (less than 0.04 s-1). A comparison of the results obtained in this study with those reported for rabbit skeletal muscle reveals qualitative similarities between cardiac and skeletal muscle and also quantitative differences in their physiological and kinetic behavior.