The fusion of mononucleate myoblast cells into multinucleate myotubes (myogenesis) has often been studied as a model system of terminal cellular differentiation. Although cell-surface changes during myogenesis have attracted much attention and a variety of surface antigens including the nicotinic cholinergic receptor, acetylcholinesterase and muscle lectins have been shown to be present on muscle cells, a detailed identification of markers specific to the various cell types has not been attempted. This is mainly because fibroblasts are a major contaminating cell type in primary muscle cultures and these cells have no known distinctive cell-surface antigen(s). For instance, surface fibronectin has been used to distinguish fibroblasts in the rat peripheral nervous system in vitro but has proved ineffective in the human muscle cell system. In addition, Lesley and Lennon found that Thy-1 antigen was present on myoblasts but not myotubes of the rat L6 muscle cell line and primary cultures. However, Thy-1 antigen is also present on rat fibroblasts. Thus, unequivocal identification of the major mononucleate cell types in muscle cultures, fibroblasts and myoblasts has not been possible. We report here the use of two surface antigens, Thy-1 and a muscle-specific antigen recognized by a monoclonal antibody, to identify unambiguously the four major types of cells present in human muscle cultures and to propose a model of cell-surface differentiation during human myogenesis.