Prolonged or unaccustomed exercise leads to muscle cell membrane damage, detectable as release of the intracellular enzyme lactic acid dehydrogenase (LDH). This is correlated to excitation-induced influx of Ca²⁺, but it cannot be excluded that mechanical stress contributes to the damage. We here explore this question using N-benzyl-p-toluene sulphonamide (BTS), which specifically blocks muscle contraction. Extensor digitorum longus muscles were prepared from 4 wk old rats and mounted on holders for isometric contractions. Muscles were stimulated intermittently at 40 Hz or exposed to the Ca²⁺ ionophore A23187. Electrical stimulation increased ⁴⁵Ca influx 3-5 fold. This was followed by a progressive release of LDH, which was correlated to the influx of Ca²⁺. BTS (50 μM) caused a 90% inhibition of contractile force, but had no effect on the excitation-induced ⁴⁵Ca influx. Following stimulation, ATP and creatine phosphate levels were higher in BTS treated muscles, probably due to the cessation of ATP-utilization for cross bridge cycling, indicating a better energy status. No release of LDH was observed in BTS treated muscles. However, when exposed to anoxia, electrical stimulation caused a marked increase in LDH release which was not suppressed by BTS, but associated with decreased content of ATP. Dynamic passive stretching caused no increase in muscle Ca²⁺ content and only a minor release of LDH, whereas treatment with A23187 markedly increased LDH release both in control and BTS treated muscles. In conclusion, following isometric contractions muscle cell membrane damage depends on Ca²⁺ influx and energy status, and not on mechanical stress.