Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).