Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 184.108.40.206) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.