These experiments examined the myosin phenotype and bioenergetic enzyme activities in rat respiratory muscles. Muscle samples were removed from adult female Sprague-Dawley rats (N = 8) and analyzed to determine the myosin heavy chain (MHC) and light chain (MLC) isoform content as well as the activities of myofibrillar ATPase (mATPase), citrate synthase (CS; Krebs cycle enzyme), and lactate dehydrogenase (LDH; glycolytic enzyme). Analysis revealed that CS activity and the % type I MHC and %IId MHC isoforms were greater in the costal diaphragm (CO-D) compared with those in the crural diaphragm (CR-D). In contrast, the % type IIb MHC was higher in the CR-D compared with that in the CO-D. LDH and mATPase activity were lower in both the CO-D and CR-D compared with that in the parasternal intercostals (PI), external intercostals (EI), internal intercostals (II), rectus abdominis (RA), and sternomastoid (SM) muscles. CS activity, % type I MHC, %IIa MHC, and the ratio of slow to total alkali MLC (1s/1s + 1f + 3f) were greater in the CO-D and CR-D compared with those in all other respiratory muscles. The RA contained the highest (P < 0.05) % type IIb MHC and lowest CS activity compared with that in all other muscles. Finally, CS activity, mATPase activity, and MHC phenotype did not differ among the PI, EI, II, and SM muscles. These differences in biochemical properties provide the muscles of the respiratory pump with great versatility in functional properties.