The molecular basis underlying the establishment of the myogenic lineage, subsequent differentiation, and the establishment of specific fiber types (i.e., fast versus slow) is becoming well understood. In contrast, the regulation of the general properties of a specific anatomical muscle group (e.g., leg versus jaw muscles) and the regulation of muscle-fiber properties within a particular group are less well characterized. We have investigated the potential role of the homeobox-containing gene, Engrailed-2 (En-2), in the mouse, which is specifically expressed in myoblasts in the first arch and maintained in the muscles of mastication in the adult. We have generated mice that ectopically express En-2 in all muscles during early development and primarily in fast muscles in the adult. Ectopic En-2 in nonjaw muscles leads to a decrease in fiber size, whereas overexpression in the jaw muscles leads to a shift in fiber metabolic properties as well as a decrease in fiber size. In contrast, loss of En-2 in the jaw leads to a shift in fiber metabolic properties in the jaw of female mice only. Jaw muscles are sexually dimorphic, and we propose that the function of En-2 and mechanisms guiding sexual dimorphism of the jaw muscles are integrated. We conclude that the specific expression of En-2 in the jaw therefore plays a role in specifying muscle-fiber characteristics that contribute to the physiologic properties of specific muscle groups.