The influence of Ca2+ dissociation rate from TnC and decreased cross-bridge detachment rate on the time course of relaxation induced by flash photolysis of diazo-2 in rabbit skinned psoas fibers was investigated at 15 degrees C. A TnC mutant (M82Q TnC) that exhibited increased Ca2+ sensitivity caused by a decreased Ca2+ dissociation rate in solution also increased the Ca2+ sensitivity of force and decreased the rate of relaxation in fibers approximately 2-fold. In contrast, a TnC mutant (NHdel TnC) with decreased Ca2+ sensitivity caused by an increased Ca2+ dissociation rate in solution decreased Ca2+ sensitivity of force but did not accelerate relaxation. Decreasing the rate of cross-bridge kinetics by reducing [Pi] slowed relaxation -2-fold and led to two phases of relaxation, a linear phase followed by an exponential phase. In fibers, M82Q TnC further slowed relaxation in low [Pi] approximately 2-fold whereas NHdel TnC had no significant effect on relaxation. These results are consistent with the interpretation that the Ca2+ dissociation rate and cross-bridge detachment rate are similar in fast twitch skeletal muscle such that decreasing either rate slows relaxation but accelerating Ca2+ dissociation has little effect on relaxation.