We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo.