The influence of substituting D2O for H2O on calcium transients and on contraction was studied in intact single skeletal muscle fibres injected with aequorin and in mechanically skinned fibres from frogs. Most experiments were carried out at 10 degrees C. Experiments performed in vitro established that the calcium concentration-effect curve for aequorin is depressed and the rate of change of aequorin light emission after sudden changes of [Ca2+] is decreased when D2O is substituted for H2O. D2O substitution greatly reduces the amplitude of the aequorin signals of intact fibres both in twitches and in tetani. This is partly the result of the influence of D2O on aequorin, but the amplitude of the calcium transients is reduced as well. In both H2O and D2O Ringer solutions, the amplitude and time course of the sarcoplasmic calcium transient during a tetanus vary greatly with the stimulus frequency. In H2O, frequencies high enough to produce mechanical fusion normally produce cytoplasmic calcium concentrations high enough to saturate the myofibrils, with the result that stimulus frequency has very little influence on the mechanical response. Saturation does not occur in D2O, and the amplitude and form of fully fused tetani are greatly influenced by the frequency of stimulation. Aequorin was used as a calcium indicator to estimate the influence of D2O substitution on the apparent equilibrium association constant for the calcium-EGTA complex. The constant was reduced by more than one order of magnitude in D2O at pD = pH = 7.0. Experiments on mechanically skinned muscle fibres showed that D2O substitution decreased the apparent sensitivity of the myofibrils to calcium, but increased the force of contraction at saturating calcium concentrations. The latter effect is probably responsible for the potentiation of tetanic tension that is observed in certain D2O/H2O mixtures or in H2O solutions immediately after a switch from D2O. In intact muscle fibres the potentiation of twitch or tetanic tension after a switch from D2O to H2O declines with a half-time of about 25 s; this probably reflects the time course of exchange of intracellular D2O for H2O. The effects of D2O on the intracellular calcium transient appeared and disappeared with a half-time of less than 1 s; this time is of the same order of magnitude as that calculated for the exchange of H2O and D2O in the T-tubules. We conclude that D2O affects calcium release at a site in the T-tubule which is more accessible to the extracellular space than to the intracellular space.