Cytochalasin B (5 μg/ml) elicits a differential effect on myoblasts and fibroblasts in culture. After 1 day in culture in the presence of the drug, two types of cells were observed, round cells and cells with elongated arms, designated “arborized” cells. Both cell types were examined in the electron microscope. The round cells contained aggregates of thin and thick filaments as well as a few intact sarcomeres. Within the arms of the arborized cells were bundles of intermediate sized filaments (100 Å in diameter). The round cells could be shaken off the culture dish, washed free of cytochalasin B, and recultured to form myotubes. The remaining arborized cells lost their stellate shape when the drug was removed. The progeny of these cells gave rise to normal fibroblasts. Cytochalasin B, thus could be used to identify and isolate myoblasts prior to their fusion into developing muscle. It is suggested that this differential effect of the drug can be used to prepare pure cultures of fusible muscle cells uncontaminated by fibroblasts.