The aim of this study was to examine the effect of the metabolites H⁺, ADP, and P_i on the rate of cardiac relaxation. We used guinea pig right ventricular trabeculae that had been chemically skinned, allowing the myofilaments to be studied in isolation. Laser-flash photolysis of the caged Ca²⁺ chelator diazo 2, causing a rapid fall in intracellular Ca²⁺, enabled investigation of relaxation independently of the rate of Ca²⁺ diffusion. On the photolysis of diazo 2, the trabeculae relaxed biphasically with exponential rate constants (k₁ and k₂) of 10.07 and 4.23 s¹, respectively, at 12°C and 18.35 and 2.52 s¹, respectively, at a nominal 20°C. Increasing the concentration of both protons (pH 7.2-6.8) and MgADP (0.5-3.4 mM) slowed the two phases of the relaxation transients. Raising the concentration of P_i from the control level of 1.36 mM to 15.2 mM increased the rate of both phases, with relaxation becoming monoexponential at 19.4 mM P_i (with a k of 20.31 s¹ at 12°C). Cardiac muscle was compared with skeletal muscle under identical conditions; in cardiac muscle 19.4 mM P_i increased the rate of relaxation, whereas in skeletal muscle this concentration of P_i slowed relaxation. We conclude that the mechanism of relaxation differs between cardiac and skeletal muscle. This study is a direct demonstration of the effects of ATP metabolites on cardiac myofilament processes during relaxation.