Some adult cranial muscles have been reported to contain unusual myosin heavy-chain (MHC) isoforms (i.e., slow-tonic, alpha-cardiac, embryonic, and neonatal), which exhibit distinct contractile properties. In this study, adult human mylohyoid (MH) muscles obtained from autopsies were investigated to detect the unusual MHC isoforms. For comparison, the biceps brachii and masseter muscles of the same subjects were also examined. Serial cross-sections from the muscles studied were incubated with a panel of isoform-specific anti-MHC monoclonal antibodies that distinguish major and unusual MHC isoforms. On average, the slow type I and fast type II MHC-containing fibers in the MH muscle accounted for 54% and 46% of the fibers, respectively. In contrast to limb and trunk muscles, the adult human MH muscle was characterized by a large proportion of hybrid fibers (85%) and a small percentage of pure fibers (15%; P < 0.01). Of the fast fiber types, the proportion of the type IIa MHC-containing fibers (92%) was much greater than that of the type IIx MHC-containing fibers (8%; P < 0.01). Our data demonstrated that the adult human MH fibers expressed the unusual MHC isoforms that were also identified in the masseter, but not in the biceps brachii. These isoforms were demonstrated by immunocytochemistry and confirmed by electrophoretic immunoblotting. Fiber-to-fiber comparisons showed that the unusual MHC isoforms were coexpressed with the major MHC isoforms (i.e., MHCI, IIa, and IIx), thus forming various major/unusual (or m/u) MHC hybrid fiber types. Interestingly, the unusual MHC isoforms were expressed in a fiber type-specific manner. The slow-tonic and alpha-cardiac MHC isoforms were coexpressed predominantly with slow type I MHC isoform, whereas the developmental MHC isoforms (i.e., embryonic and neonatal) coexisted primarily with fast type IIa MHC isoform. There were no MH fibers that expressed exclusively unusual MHC isoforms. Approximately 81% of the slow type I MHC-containing fibers expressed slow-tonic and alpha-cardiac MHC isoforms, whereas 80% of the fast type IIa MHC-containing fibers expressed neonatal MHC isoform. The m/u hybrid fibers (82% of the total fiber population) were found to constitute the predominant fiber types in the adult human MH muscle. At least seven m/u MHC hybrid fiber types were identified in the adult human MH muscle. The most common m/u hybrid fiber types were found to be the MHCI/slow-tonic/alpha-cardiac and MHCIIa/neonatal, which accounted for 39% and 33% of the total fiber population, respectively. The multiplicity of MHC isoforms in the adult MH fibers is believed to be related to embryonic origin, innervation pattern, and unique functional requirements.