Summary1. The effect of short‐term, reversible alteration of muscle total creatine content (Crtot) on force development was sought in fast‐twitch extensor digitorum longus (EDL) muscles of female mice.2. Three in vivo interventions were investigated: 1% creatine‐supplementation, treatment with the creatine‐uptake inhibitor β‐guanidino propionic acid (β‐GPA; 1%) or β‐GPA treatment followed by creatine supplementation for 5 days.3. The Crtot of isolated muscles, determined using reverse‐phase high‐performance liquid chromatography, was 133 ± 38 mmol/kgdry in 11 EDL control muscles and was not significantly affected by dietary creatine‐supplementation (152 ± 25 mmol/kgdry; n = 8). Significant creatine depletion was observed in the β‐GPA‐fed group (65 ± 6 mmol/kgdry; n = 9) and this was reversed by 5 days of creatine supplementation (133 ± 21 mmol/kgdry; n = 10).4. Creatine depletion did not affect maximum tetanic stress. However, when muscle creatine was restored by creatine supplementation, a substantial increase in tetanic stress was observed. Significant correlations were observed between Crtot and maximum tetanic stress (r = 0.56) and relaxation rate (r = 0.43). The enhancement of force was not due to effects of creatine on muscle fibre type because neither mechanical tests of fibre characteristics nor the fibre types of the muscles were affected.5. We conclude that, in muscles that contain large numbers of fast‐twitch fibres, maximum tetanic stress is determined, in part, by muscle creatine stores.