Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the 'slow' area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age.(ABSTRACT TRUNCATED AT 250 WORDS)