The accumulation of creatine phosphokinase (CPK) activity during the development of quail (C. coturnix japonica) myoblasts in culture was determined under both fusion permissive and impermissive (low Ca2+) conditions. When fusion is prevented by decreasing the [Ca2+] of growth restrictive medium, accumulation is initiated at the same time, follows the same kinetics and attains the same steady-state level as that in muscle fibers which form in the same medium with the normal level of Ca2+. Individual clones, in which complete homogeneity of cell type can be assured, were employed to establish levels of CPK activity per nucleus at the prefusion stage and in clones in which every nucleus is included within a syncytium. Cross-comparisons were made between these values and the activity measurements derived from mass cultures. These comparisons indicate no significant effect of fibroblast contamination or of myogenic cells of more advanced or retarded stages on the sign or magnitude of increase in CPK activity. Based on the parameters measured and the qualitative evidence that the same isozyme shift occurs in myocytes as in muscle fibers there is no reason to assume that differentiation is less complete or regulated differently in fusion-blocked myocytes. These studies indicate the utility and justification of employing this cell system and the biochemical criterion employed to examine myogenic differentiation on a cell by cell basis.