In vivo and in vitro, proliferating motile myoblasts form aligned groups of cells, with a characteristic bipolar morphology, subsequently become post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members of the myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin isoforms, from at least five different classes of the myosin superfamily (classes I, II, V, VII and IX), using RT-PCR and degenerate primers to conserved regions of myosin. All of these myosin isoforms were expressed most highly in myoblasts and their expression decreased as they differentiated into mature myotubes, by RNAse protection assays, and Western analysis. However, only myosin I alpha, non-muscle myosin IIA and IIB together with actin relocalize in response to the differentiative state of the cell. In single cells, myosin I alpha was found at the leading edge, in rear microspikes and had a punctate cytoplasmic staining, and non-muscle myosin was associated with actin bundles as previously described for fibroblasts. In aligned groups of cells, all these proteins were found at the plasma membrane. Co-staining for skeletal myosin II, and myosin I alpha showed that myosin I alpha also appeared to be expressed at higher levels in post-mitotic myoblasts that had begun to express skeletal myosin prior to fusion. In early myotubes, actin and non-muscle myosin IIA and IIB remained localized at the membrane. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform of myosin I (mouse homologue to myr2) showed a punctate cytoplasmic staining which did not change as the myoblasts differentiated. In conclusion, although we found that myoblasts express many different isoforms of the myosin superfamily, only myosin I alpha, non-muscle myosin IIA and IIB appear to play any direct role in myogenesis.