1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26.1 +/- 1.5 and 26.1 +/- 1.3 nC microF-1, mid-point voltage of -35.1 +/- 5.0 and -42.2 +/- 1.2 mV and steepness of 16.7 +/- 2.2 and 17.0 +/- 1.9 mV (means +/- S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 +/- 18 and 115 +/- 8 nM (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 +/- 1.0 microM; n = 7), than in guinea-pigs (2.8 +/- 0.3; n = 4). 4. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 +/- 0.1 and 0.9 +/- 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 +/- 0.08 and 0.81 +/- 0.06% ms-1 in rats and 2.43 +/- 0.25 and 0.88 +/- 0.01% ms-1 in guinea-pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5. These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.