This study utilized N-benzyl-p-toluene sulfonamide (BTS), a potent inhibitor of crossbridge cycling, to measure 1) the relative metabolic costs of crossbridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca²⁺ ([Ca²⁺]_c) or intracellular PO₂ (P_iO₂) were measured during contractions at 20°C in control conditions (CON) and following exposure to 12.5µM BTS. BTS attenuated tension development to 5% ± 0.4% of CON but did not affect either resting or peak [Ca²⁺]_c during repeated isometric contractions. To determine the relative metabolic cost of crossbridge cycling, the fall in P_iO₂ (P_iO₂; a proxy for VO₂) during contractions was measured in CON and BTS. BTS attenuated P_iO₂ by 55% ± 6%, reflecting the relative ATP cost of crossbridge cycling. Thus, extrapolating P_iO₂ to that which would occur at 0% tension suggests that actomyosin ATP requirement is ~58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in P_iO₂ (time to 63% of overall P_iO₂) from 75 ± 9 sec (CON) to 101 ± 9 sec (BTS) (p<0.05), suggesting an important role of the products of ATP hydrolysis in determining the VO₂ onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (~42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca²⁺]_c transients, a reduced rate of ATP consumption resulted in slower VO₂ onset kinetics.