Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was used as an intracellular Ca2+ chelator to shorten the duration of the Ca2+ transient and to determine the rate-limiting steps in relaxation of frog skeletal muscle at 10 and 20 degrees C. Incubation with the acetoxymethyl ester of EGTA (EGTA-AM) produced a linear approximately twofold increase in the rate of fall in twitch Ca2+ concentration ([Ca2+]) over 0-70 min at 10 degrees C. The rate of relaxation initially increased from 5 to 9/s over 0-13 min and then leveled, as the rate of fall in [Ca2+] continued to increase and peak force decreased. Increasing the rate of fall in [Ca2+] increased the rate of relaxation, until a rate-limiting step was reached at approximately 9/s at 10 degrees C. At 20 degrees C, incubation with EGTA-AM produced a linear approximately twofold increase in the rate of fall in twitch [Ca2+] without affecting the rate of relaxation (27/s). Ca2+ dissociated from the regulatory sites of purified troponin (Tn) at approximately 8/s at 10 degrees C and at 23/s at 20 degrees C. When the duration of the Ca2+ transient was decreased by EGTA or increased by partial inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase, twitch force increased linearly with Ca2+ transient duration. Thus the duration of the Ca2+ transient is a primary determinant of twitch force and, whereas the rate of fall in [Ca2+] is rate limiting for relaxation at 10 degrees C, Ca2+ dissociation from Tn may be rate limiting at 20 degrees C.