In this study we investigated the roles of cytoplasmic ATP as both an energy source and a regulatory molecule in various steps of the excitation–contraction (E–C) coupling process in fast‐twitch skeletal muscle fibres of the rat. Using mechanically skinned fibres with functional E–C coupling, it was possible to independently alter cytoplasmic [ATP] and free [Mg²⁺]. Electrical field stimulation was used to elicit action potentials (APs) within the sealed transverse tubular (T‐) system, producing either twitch or tetanic (50 Hz) force responses. Measurements were also made of the amount of Ca²⁺ released by an AP in different cytoplasmic conditions. The rate of force development and relaxation of the contractile apparatus was measured using rapid step changes in [Ca²⁺]. Twitch force decreased substantially (∼30%) at 2 mm ATP compared to the level at 8 mm ATP, whereas peak tetanic force only declined by ∼10% at 0.5 mm ATP. The rate of force development of the twitch and tetanus was slowed only slightly at [ATP]≥ 0.5 mm, but was slowed greatly (> 6‐fold) at 0.1 mm ATP, the latter being due primarily to slowing of force development by the contractile apparatus. AP‐induced Ca²⁺ release was decreased by ∼10 and 20% at 1 and 0.5 mm ATP, respectively, and by ∼40% by raising the [Mg²⁺] to 3 mm. Adenosine inhibited Ca²⁺ release and twitch responses in a manner consistent with its action as a competitive weak agonist for the ATP regulatory site on the ryanodine receptor (RyR). These findings show that (a) ATP is a limiting factor for normal voltage‐sensor activation of the RyRs, and (b) large reductions in cytoplasmic [ATP], and concomitant elevation of [Mg²⁺], substantially inhibit E–C coupling and possibly contribute to muscle fatigue in fast‐twitch fibres in some circumstances.