Myoblasts and myotubes are heterogeneous, but what is the significance of this heterogeneity? Is it a vital component of the mechanism by which a muscle develops or is it part of the process that generates mature fibers with diverse sizes, speeds of contracture, and metabolisms? We have begun to explore these questions by using BrdU to selectively label rat primary myotubes, thus enabling their mature characteristics to be defined for the first time. In the soleus, the type I fibers of primary myotube origin were 21% larger than those of secondary myotube origin, indicating that the origin of a fiber can affect its mature force production. In the extensor digitorum longus (EDL), the primary myotubes differentiated into all known fibers types, but with marked variation in frequency. In the superficial portion of the EDL, 97% of primary myotubes became IIB fibers, even though approximately 41% of the fibers in this region are IIA or IIX. In the deep portion, primary myotubes preferentially developed into type I fibers. Thus, primary myotubes in the EDL predominantly differentiate into the two most dissimilar fiber types: the slowest, smallest, most oxidative, type I fibers and the largest, fastest, most glycolytic, type IIB fibers. Each of the subtypes of primary myotubes had a different fate. In the EDL, the slow and fast primary myotubes appeared to differentiate into type I and IIB fibers, respectively. This implies that spatial and temporal signals operating in the limb are major determinants of the mature pattern of fiber types and that innervation of a muscle involves a selective matching between the various types of motoneurons and muscle fibers.