The chicken as a research model has a disadvantage compared with the mouse and the human because of the low number of available antibodies against gene products of interest. The goal of this study was to identify the antigen recognized by monoclonal antibody (mAb) GIIF3, which is a 42 kDa protein that appears in follicle-associated epithelium of the guinea hen as well as in different muscle types during chicken embryonic development. The 42 kDa protein, immunoprecipitated from chicken gizzard protein lysates, was evaluated by mass spectrometry. Mass spectrometry analysis revealed peptides specific for the chicken β- or γ-actin isoforms. The mAb GIIF3 can be used as a new research tool for smooth muscle cell and bursa of Fabricius developmental studies.