A beta-D-galactoside binding lectin has been proposed to play a role in the cell interactions required for skeletal myogenesis. However, conflicting results have challenged this model as the basis for myoblast interactions and fusion. We have studied the effects of thiodigalactoside on the differentiation of the myogenic L8 line of rat cells and on myoblasts from newborn rats. Our results do not support a role for this lectin in the interactions of myoblasts immediately preceding fusion or in myoblast fusion per se. Thiodigalactoside did not inhibit development when present for restricted periods preceding fusion; differentiation was delayed only when cells were grown in the sugar for prolonged periods. Although a thiodigalactoside binding lectin is present in extracts of developing myoblasts, it is also present in equivalent amounts in developmentally defective nonfusing variants. Using antibody specific for this lectin and indirect immunofluorescence, myoblasts could be distinguished from fibroblasts; however, most of the lectin-associated antigen was within and not on the cells. One pattern of distribution of lectin determinants, similar to that reported with antiactin antibody, was present in differentiating L8 cells but not in the transformed fu-1 variant.