Ultrastructural and physiological studies have shown that planarian muscles have some characteristics of smooth and some characteristics of striated muscles. To characterize planarian muscles, we isolated two myosin heavy chain genes (DjMHC-A and DjMHC-B) from a planarian, Dugesia japonica, by immunological screening, and analyzed their structures and spatial expression patterns. Structural analysis indicated that both MHC genes are striated-muscle-type myosin genes, although planarian muscles do not have any striation. In situ RNA hybridization showed that expression of the two myosin genes is spatially strictly segregated. DjMHC-A was expressed in pharynx muscles, pharynx cavity muscles, muscles surrounding the intestinal ducts, a subpopulation of body-wall muscles and several muscle cells in the mesenchymal region around the base of the pharynx. DjMHC-B was expressed in body-wall muscles (including circular, diagonal and longitudinal muscles), vertical muscles and horizontally oriented muscles. Double staining with DjMHC-A and -B probes clearly demonstrated that expression of the DjMHC-A and -B genes do not occur in the same cell. During regeneration, the number of cells positive for expression of each gene increased in the blastema region, suggesting that both types of muscle may be involved in blastema formation. DjMHC-B-positive cells disappeared from the body-wall muscle layer in the pharynx-cavity-forming region, whereas DjMHC-A-positive cells were markedly accumulated there, suggesting that the two types of muscle in the body wall layer may have distinct functions. These results indicate that planarians have at least two types of muscle that express striated-muscle-type MHC genes, but do not form striation.