S46, a monoclonal antibody (mAb) specific for the SM-1 and SM-2 isoforms of avian slow myosin heavy chains (MHC), was used to study the earliest stages of development of intrafusal fibers in muscle spindles of the rat hindlimb. Spindles formed only in the regions of fetal muscles that contained primary myotubes reactive to mAb S46, such as the axial region of the tibialis anterior muscle. The first intrafusal fiber to form, the nuclear bag2 fiber, originated from within the population of S46-reactive primary myotubes. Binding of mAb S46 by myotubes giving rise to the bag2 fibers preceded the appearance of encapsulated spindles in the muscles by electron microscopy. However, reactivity to S46 intensified in the myotubes transforming into bag2 fibers after the innervation of the fibers by afferents, and dissipated in myotubes differentiating into slow-twitch (type I) extrafusal fibers. Thus, afferents may enhance intrafusal expression of the MHC isoform reactive to mAb S46. The pattern of S46 binding to nuclear bag and chain intrafusal fibers in both developing and adult spindles was the same as that reported for the mAb ALD19, suggesting that both antibodies bind to the same MHC isoform. This isoform is probably a developmental form of slow myosin, because it was transiently expressed during the development of type I extrafusal fibers. The origin of bag2 intrafusal and type I extrafusal fibers from a bipotential subpopulation of primary myotubes reactive to mAb S46 correlates with the location of muscle spindles in the slow regions of muscles in adult rat hindlimbs.