6-Mercaptopurine (6-MP) is an adenine antagonist which has been shown to cause skeletal muscle atrophy in neonatal rats. To investigate the effects of 6-MP on developing muscles, pure populations of myoblasts and myotubes in vitro were treated with 6-MP (5-50 micrograms/ml). The viability and protein content of myotubes, but not myoblasts, was decreased by 6-MP. Microscopic examination of the 6-MP-treated (50 micrograms/ml) myotubes demonstrated that severe degenerative changes had occurred, including extensive necrosis, inhibition of myotube formation, and the appearance of intracellular vacuoles. However, 6-MP treatment decreased the incorporation of radiolabeled leucine and thymidine by myoblasts, but not myotubes. These findings indicate that 6-MP exerted a selective toxicity on myotubes but not myoblasts.