In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.