We studied serial sections of human fetal limb muscles (10-25 weeks of gestation) by light microscopic (LM) immunocytochemistry, using specific antibodies against slow-tonic, slow-twitch, fetal, embryonic, and alpha-cardiac myosin heavy chain (MHC) isoforms, neurofilament protein, laminin, and myomesin. One set of the first-generation myotubes expressed slow-tonic MHCs, and slow-twitch, fetal, and embryonic MHCs from the tenth week of gestation. These primary myotubes were identified as developing nuclear bag fibers. Second-generation myotubes in close apposition to the primary nuclear bag myotubes initially expressed only fetal and embryonic MHCs. One or more of these secondary myotubes acquired expression of slow-tonic and slow-twitch MHCs and gave rise to nuclear bag fibers. Most of the nuclear bag precursors expressed alpha-cardiac MHC. The secondary myotubes that expressed fetal and embryonic MHC but not slow-tonic, slow-twitch, or alpha-cardiac MHCs gave rise to the nuclear chain fibers. This study shows that different populations of fiber precursors, each with a unique sequence of MHC expression, gave rise to the nuclear bag and chain fibers, despite the presence of a common afferent nerve, from the early stages.