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Avian thymic hormone (ATH), a parvalbumin with immunomodulating properties, was used as antigen for development of a hybridoma cell line. Hybridoma screening by enzyme-linked immunosorbent assay (ELISA) led to the culture, purification, and characterization of an hybridoma, designated 4A6, which secretes IgG1 that is specific for ATH. The monoclonal antibody (MAb) was characterized by techniques involving indirect competitive and non-competitive ELISA, Western Blot analysis, and immunohistological staining. The MAb was highly specific for ATH with no cross-reactivity to other chicken tissue extracts or commercially available parvalbumins.