Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC_2X isoform but not in fibers expressing MHC_Slow and MHC_2A (10, 12). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP_iso) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC_2X. In single type-identified, permeabilized DIAm fibers, ATP_iso was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (Vmax ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V_o) and cross bridge cycling rate (estimated from the rate constant for force redevelopment (k_TR) following quick release and restretch) was also examined. Two weeks after DNV, ATP_iso was significantly reduced in fibers expressing MHC_2X, but unaffected in fibers expressing MHC_Slow and MHC_2A. This effect of DNV on fibers expressing MHC2X persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V_o and k_TR were slowed in fibers expressing MHC_2X, consistent with the effect on ATP_iso. The difference between V_max ATPase and ATP_iso reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC_2X. DNV-induced reductions in ATP_iso and V_max ATPase of fibers expressing MHC_2X reflect the underlying decrease in MHC content, while reduction in ATP_iso also reflects a slowing of cross bridge cycling rate.