The effect of exogenous hydrogen peroxide (H₂O₂) on excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function was compared in mechanically skinned slow twitch fibres (prepared from the soleus muscles) and fast twitch fibres (prepared from the extensor digitorum longus; EDL muscles) of adult rats. Equilibration (5 min) with 1 mm H₂O₂ diminished the ability of the Ca²⁺-depleted SR to reload Ca²⁺ in both slow (P < 0.01) and fast twitch fibres (P < 0.05) compared to control. Under conditions when all Ca²⁺ uptake was prevented, 1 mm H₂O₂ increased SR Ca²⁺ 'leak' in fast twitch fibres by 24 ± 5 % (P < 0.05), but leak was not altered in slow twitch fibres. Treatment with 1 mm H₂O₂ also increased the peak force of low [caffeine] contracture by ~45 % in both fibre types compared to control (P < 0.01), which could be partly reversed following treatment with 10 mm dithiothreitol (DTT). The changes in SR function caused by 1 mm H₂O₂ were associated with an ~65 % increase in the peak height of depolarization-induced contractile response (DICR) in slow twitch fibres, compared to control (no H₂O₂; P < 0.05). In contrast, peak contractile force of fast twitch fibres was not altered by 1 mm H₂O₂ treatment. Equilibration with 5 mm H₂O₂ induced a spontaneous force response in both slow and fast twitch fibres, which could be partly reversed by 2 min treatment with 10 mm DTT. Peak DICR was also increased ~40 % by 5 mm H₂O₂ in slow twitch fibres compared to control (no H₂O₂; P < 0.05). Our results indicate that exogenous H₂O₂ increases depolarization-induced contraction of mechanically skinned slow but not fast twitch fibres. The increase in depolarization-induced contraction in slow twitch fibres might be mediated by an increased SR Ca²⁺ release during contraction and/or an increase in Ca²⁺ sensitivity.