C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, delta isoform (PP1c delta). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1c delta was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1c delta, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.