Ca2+ and Mg2+ movements across the sarcoplasmic reticulum (SR) of frog skeletal muscle fibers were measured in situ by electron probe microanalysis of muscles rapidly frozen following a tetanus. At 400 ms following a 1.2-s tetanus at room temperature, the force had relaxed to base-line, and 0.3 mmol of Ca2+/liter of cytoplasmic H2O had been pumped by the SR, indicating that the in situ pumping of the SR Ca-ATPase is sufficiently high to account for the removal of Ca2+ from the Ca2+-specific sites of troponin (0.18 mmol of Ca2+-specific sites/liter of cytoplasmic H2O) and for the rate of relaxation from a tetanus at room temperature. The half-time of the return of the total 1.0 mmol of Ca2+/liter of cytoplasmic H2O released during a tetanus was 1.1 s, comparable to the slow Koff rate of Ca2+ from (carp) parvalbumin (1.0 s-1) and consistent with the hypothesis that the return of this Ca2+ to the terminal cisternae is rate-limited by the Ca2+ off-rate from parvalbumin. The return of the Mg2+ taken up by the terminal cisternae during a tetanus to resting levels was significantly slower than the time course of the Ca2+ movements, suggesting that the Mg2+ permeability of the SR in situ is low and may be transiently increased during tetanic stimulation.