1. The effect of rapid shortening on rate of force regeneration (dF/dtR) was examined in single, intact frog (Rana temporaria) skeletal muscle fibres (3.0 C). Step releases leading to unloaded shortening were applied after 500 ms of stimulation, during the plateau of an isometric tetanus. Initial mean sarcomere length ranged from 2.05 to 2.35 micrometer; force regeneration after shortening was at 2.00 micrometer. 2. Values for dF/dtR following a 25 nm half-sarcomere-1 release were 3.17 +/- 0.17 (mean +/- s.e.m., n = 8) times greater than the initial rate of rise of force before release (dF/dtI). As release size was increased from 25 to 175 nm half-sarcomere-1, the relationship between release size and dF/dtR decreased sharply before attaining a plateau value that was 1.34 +/- 0.09 times greater than dF/dtI. Despite wide variations in dF/dtR, the velocity of unloaded shortening remained constant (2.92 +/- 0.08 micrometer half-sarcomere-1 s-1; n = 8) for the different release amplitudes used in this study. 3. To investigate its role in the attenuation of dF/dtR with increased shortening, the effects of rapid ramp (constant velocity) shortening on intracellular free Ca2+ concentration ([Ca2+]i) were monitored using the Ca2+-sensitive fluorescent dye furaptra. Compared with an isometric contraction, rapid fibre shortening was associated with a transient increase in [Ca2+]i while force regeneration after shortening was associated with a transient reduction in [Ca2+]i. The greatest reductions in [Ca2+]i were associated with the largest amplitude ramps. 4. Cross-bridge-mediated modifications of the Ca2+ affinity of troponin C (TnC) may explain the fluctuations in [Ca2+]i observed during and after ramps. Associated fluctuations in TnC Ca2+ occupancy could play a role in the reduction of dF/dtR with increasing release size.