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Adenovirus (Ad) has been used in vivo and in vitro as a vector to carry a foreign gene for efficient gene delivery into various cell types and tissues of animals. The aim of the current study was to evaluate the Ad delivery system in primary avian cells. Primary cells isolated from the embryonic pectoralis major muscles of the chicken and quail were cultured and incubated with human recombinant Ad serotype 5 (Ad5) containing sequences encoding either the green fluorescence protein (GFP) gene alone, as a tracking marker, or both GFP and murine 3-hydroxyisobutyryl-CoA hydrolase (mHIBCH) as a target gene. The fluorescent GFP images showed the successful delivery of a target gene using Ad5 in the primary avian cultured cells. In addition, immunostaining of the myosin heavy chain (MyHC) in these cells indicated that a large population of the cells was myogenic. Colocalization of GFP-positive cells with MyHC staining was mostly found in MyHC-negative cells, indicating successful delivery of Ad5 into a large population of mononucleated cells. Furthermore, the current fluorescence study detected the dual expression of GFP and mHIBCH protein in GFP-positive cells. Finally, Western blot analysis confirmed that the Ad-mediated expression of mHIBCH protein was specific in primary cultures of avian myogenic cells and that the mHIBCH protein expression was continued for 15 d after infection in chicken primary cells. These data demonstrate that Ad5 is a feasible tool to express foreign genes in primary cultured cells of avian species, providing a new approach to study the function of genes of interest in muscle development and metabolism.