1. The Ca2+ content of single mammalian skeletal muscle fibres was determined using a novel technique. Mechanically skinned fibres were equilibrated with varying amounts of the Ca2+ buffer BAPTA and were then lysed in a detergent-paraffin oil emulsion. The subsequent myofilament force response was used to estimate the additional amount of Ca2+ bound to BAPTA following lysis of intracellular membranes. 2. The total endogenous Ca2+ content (corrected for endogenous Ca2+ buffering) of fast-twitch (FT) and slow-twitch (ST) fibres at a myoplasmic pCa (-log[Ca2+]) of 7.15 was 1.32 +/- 0.02 and 1.35 +/- 0.08 mM per fibre volume, respectively. The sarcoplasmic reticulum (SR) component of these estimates was calculated as 1.01 and 1.14 mM, respectively, which normalized to SR volume corresponds to resting SR Ca2+ contents of 11 and 21 mM, respectively. 3. Equilibration of 'resting' fibres with low myoplasmic [Ca2+] (pCa 7.67-9.00) elicited a time-dependent decrease in Ca2+ content in both fibre types. Equilibration of resting fibres with higher myoplasmic [Ca2+] (pCa 5.96-6.32) had no effect on the Ca2+ content of ST fibres but increased the Ca2+ content of FT fibres. The maximum steady-state total Ca2+ content (3.85 mM) was achieved in FT fibres after 3 min equilibration at pCa 5.96. Equilibration at higher myoplasmic [Ca2+] was less effective, probably due to Ca2(+)-induced Ca2+ release from the SR. 4. Exposure of fibres to either caffeine (30 mM, pCa approximately 8, 2 min) or low myoplasmic [Mg2+] (0.05 mM, pCa approximately 9, 1 min) released approximately 85% of the resting SR Ca2+ content. The ability of caffeine to release SR Ca2+ was dependent on the myoplasmic Ca2+ buffering conditions. 5. The results demonstrate that the SR of ST fibres is saturated with Ca2+ at resting myoplasmic [Ca2+] while the SR of FT fibres is only about one-third saturated with Ca2+ under equivalent conditions. These differences suggest that the rate of SR Ca2+ uptake in FT fibres is predominantly controlled by myoplasmic [Ca2+] while that of ST fibres is more likely to be limited by the [Ca2+] within the SR lumen.