Although parvalbumins are generally viewed as intracellular Ca2- buffers/transporters, avian species express two thymus-specific isoforms that may play alternative biological roles. These proteins, known as avian thymic hormone (ATH) and chicken parvalbumin 3 (CPV3), are conjectured to influence thymopoiesis. In this paper, we compare their intrathymic distributions. Isoform-specific monoclonal antibodies against ATH and CPV3 were labeled with fluorescein and Cy5, respectively, and then used to probe paraffin sections of chicken thymus tissue. Confocal microscopy of the stained sections reveals that ATH and CPV3 are both confined to the thymic cortex and that they are frequently coexpressed by a subset of epithelial cells. However, their expression patterns are not completely superimposible. Cortical epithelial cells are also observed that stain for just one of the two avian parvalbumin isoforms, albeit at lower frequency. Significantly, a subset of cortical thymocytes exhibits peripheral staining for one or both of the proteins. This result may imply the existence of plasma membrane receptors for the two proteins on select T-cell precursors. Alternatively, it may signal low level expression of the thymic parvalbumins by the thymocytes population.