The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (P_i) and H⁺ ([MgADP], [Pi] and [H⁺], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k₁ [5.6±0.1 s^–1 for barnacle, n=30; 26.3±0.7 s^–1 for frog, n=14 (mean±SEM)] and k₂ [0.6±0.1 s^–1 in barnacle, n=30; 10.4±1.0 s^–1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k₁ for barnacle relaxation [5.6±0.1 s^–1 (mean±SEM), n=15] compared to the decrease in k₁ of 40% seen in frog. Use of the Ca²⁺-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa₅₀ value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in k_off from the labelled protein [322±32 s^–1 at pH 7.0 and 381±24 s^–1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 µM (50 µM added ADP) from control values of 50 µM in frog decreased k₁ to 12.3±0.4 s^–1 (mean±SEM, n=8), and at 400 µM MgADP, k₁=9.6±0.1 s^–1 (mean±SEM, n=12). In barnacle, 500 µM MgADP had a much smaller effect on k₁ (4.0±0.9 s^–1, mean±SEM, n=8). Increasing the free [P_i] from the contaminant level of 0.36 mM to 1.9 mM slowed k₁ by ≈15% in barnacle [4.8±0.8 s^–1, mean±SEM, n=7], compared to a ≈30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation.