In the present study, we tested the hypothesis that intrinsic differences in ATP consumption rate per cross bridge exist across rat diaphragm muscle (Dia_m) fibers expressing different myosin heavy chain (MHC) isoforms. During maximum Ca²⁺ activation (pCa 4.0) of single, Triton X-permeabilized Dia_m fibers, isometric ATP consumption rate was determined by using an NADH-linked fluorometric technique. The MHC concentration in single Dia_m fibers was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. Isometric ATP consumption rate varied across Dia_m fibers expressing different MHC isoforms, being highest in fibers expressing MHC_2X (1.14 ± 0.08 nmol · mm³ · s¹) and/or MHC_2B (1.33 ± 0.08 nmol · mm³ · s¹), followed by fibers expressing MHC_2A (0.77 ± 0.11 nmol · mm³ · s¹) and MHC_Slow (0.46 ± 0.03 nmol · mm³ · s¹). These differences in ATP consumption rate also persisted when it was normalized for MHC concentration in single Dia_m fibers. Normalized ATP consumption rate for MHC concentration varied across Dia_m fibers expressing different MHC isoforms, being highest in fibers expressing MHC_2X (2.02 ± 0.19 s¹) and/or MHC_2B (2.64 ± 0.15 s¹), followed by fibers expressing MHC_2A (1.57 ± 0.16 s¹) and MHC_Slow (0.77 ± 0.05 s¹). On the basis of these results, we conclude that there are intrinsic differences in ATP consumption rate per cross bridge in Dia_m fibers expressing MHC isoforms.