Whether any fibers in rat hindlimb muscles express alpha-cardiac myosin heavy chain (MHC) is uncertain. Expression of alpha-cardiac MHC mRNA and the polypeptide for which it codes were examined in control and denervated rat muscle spindles using in situ hybridization (ISH) and immunocytochemistry (ICC). Both nuclear bag2 and bag1 intrafusal fibers in the extensor digitorum longus (EDL) muscles expressed alpha-cardiac MHC and its precursor mRNA. Furthermore, denervation of the hindlimb down-regulated alpha-cardiac MHC mRNA expression in rat nuclear bag intrafusal fibers, even though they continued to display a strong affinity for anti-alpha-cardiac MHC monoclonal antibody. These data show that (1) intrafusal fibers express the alpha-cardiac MHC gene; (2) innervation regulates alpha-cardiac MHC gene expression at a pre-translational level; and (3) ISH is more sensitive than ICC to changes in alpha-cardiac MHC gene expression in adult rat muscle spindles.