Experiments were done to test the hypothesis that parvalbumin (PA) promotes relaxation in frog skeletal muscle. Single fibers and purified PA from R. temporaria skeletal muscle were used to determine the relationship between Ca2+ and Mg2+ dissociation rates from PA and changes in relaxation rate as a function of isometric tetanus duration at 0 degrees C. Relaxation rate slows as a function of tetanus duration with a rate constant of 1.18 s-1. Recovery of relaxation rate after a prolonged tetanus exhibits a rate constant of 0.12 s-1. Dissociation rate constants for Mg2+ and Ca2+ from purified PA are 0.93 s-1 and 0.19 s-1, respectively. Thus rates of slowing and recovery of relaxation rate may be controlled by Mg2+ and Ca2+ dissociation from PA, respectively. The influence of temperature on relaxation rate and on Ca2+ and Mg2+ dissociation rates from purified PA also was examined. The magnitude of slowing of relaxation rate with increasing tetanus duration, relative to the final, steady value of relaxation rate, is greater at 0 than at 10 degrees C. In the 0 to 10 degrees C range, the Q10 for relaxation rate increases with increasing tetanus duration. Both of these observations can be explained if the Q10 for Ca2+ uptake by the sarcoplasmic reticulum is greater than the Q10 for Ca2+ sequestration by PA during relaxation. When Ca2+ and Mg2+ dissociation rates from PA at various temperatures are compared to other proposed indicators of PA function, it is concluded that PA facilitates relaxation of frog skeletal muscle in the 0 to 20 degrees C range.