Creatine phosphate (CP) and creatine kinase (CK) are involved in the rapid resynthesis of ATP and thereby serve to stabilize ATP concentration and to maintain free ADP low inside cardiac muscle cells during contraction. Recently, it has been suggested from experiments in permeabilized multicellular preparations that CP/ CK also regulate the kinetics of the actomyosin interaction (cross-bridge cycle) and may explain contractile dysfunction, for instance, during ischemia. However, the reported effects of CP/CK may be confounded by diffusion limitations in multicellular preparations in which inorganic phosphate (Pi) and ADP may significantly accumulate during contraction. To test this hypothesis, we measured force production and the rates of force development (AACT and &TR) in isolated cardiac myofibrils, in which rapid concentration changes of Ca2+, CP/CK, and P1 were imposed using a rapid perfusion change system. The results showed that CP/CK did not influence maximum force-generating capacity, whereas Pi markedly reduced force and increased the rates of force development. No effects of CP/CK on the rates of force development were observed, consistent with the notion that CP/CK do not exert a direct effect on the actomyosin interaction.