A stable, temperature-sensitive, nonfusing variant of the L6 rat myoblast cell line has been isolated following mild EMS-induced mutagenesis. At the permissive temperature (37° C), the growth characteristics and developmental pattern of the tsA1 variant are essentially identical to those of the parental L6D0 line at either 37° C or 40° C. At the nonpermissive temperature (40° C), the tsA1 variant grows normally but does not align, fuse, or synthesize detectable amounts of β-tropomyosin or myosin LC2. A peptide corresponding to myosin LClemb is barely detectable. The temperature-sensitive period spans the interval from 4 to 72 h postplating with a midpoint at approximately 40 h. Under standard culture conditions, commitment to terminal differentiation occurs between days 3 and 4, and alignment and fusion begin on days 4 and 5, respectively. Thus, the temperature-sensitive event occurs very early in the L6 developmental program. A spontaneous revertant of the temperature-sensitive phenotype (tsA1[R3]) exhibits recovery of the capacities to align, fuse, and synthesize the repertoire of muscle-specific proteins, suggesting that a single pleiotropic mutation in the tsA1 variant may regulate several stages in L6 myogenesis.