Aim: Force generation and transmembrane ion pumping account for the majority of energy expended by contracting skeletal muscles. Energy turnover for ion pumping, activation energy turnover (EA), can be determined by measuring the energy turnover when force generation has been inhibited. Most measurements show that activation accounts for 25-40% of isometric energy turnover. It was recently reported that when force generation in mouse fast-twitch muscle was inhibited using N-benzyl-p-toluenesulphonamide (BTS), activation accounted for as much as 80% of total energy turnover during submaximal contractions. The purpose of this study was to compare EA measured by inhibiting force generation by: (1) the conventional method of reducing contractile filament overlap; and (2) pharmacological inhibition using BTS. Methods: Experiments were performed in vitro using bundles of fibres from mouse fast-twitch extensor digitorum longus (EDL) muscle. Energy turnover was quantified by measuring the heat produced during 1-s maximal and submaximal tetanic contractions at 20 and 30 C. Results: EA measured using reduced filament overlap was 0.36 0.04 (n = 8) at 20 C and 0.31 0.05 (n = 6) at 30 C. The corresponding values measured using BTS in maximal contractions were 0.46 0.06 and 0.38 0.06 (n = 6 in both cases). There were no significant differences among these values. EA was also no different when measured using BTS in submaximal contractions. Conclusion: Activation energy turnover is the same whether measured using BTS or reduced filament overlap and accounts for slightly more than onethird of isometric energy turnover in mouse EDL muscle.