The pineal gland of viscacha exhibits histophysiological variations throughout the year, with periods of maximal activity in winter and minimal activity in summer. The aim of this work is to analyze the interstitial cells (IC) in the pineal gland of male viscachas in relation to season and age. The S-100 protein, glio-fibrillary acidic protein (GFAP), and vimentin were detected in adult and immature animals by immunohistochemistry (IHC). Double-IHC was also performed. The S-100 protein was localized within both, IC nucleus and cytoplasm. GFAP was present only in the cytoplasm. Vimentin was expressed in some IC, besides endothelial cells, and perivascular spaces. In the adult males, the morphometric parameters analyzed for the S-100 protein and GFAP exhibited seasonal variations with higher values of immunopositive area percentage in winter and lower values in summer, whereas the immature ones showed the lowest values for all the adult animals studied. Colocalization of S-100 protein and GFAP was observed. The IC exhibited differential expression for the proteins studied, supporting the hypothesis of the neuroectodermal origin. The IC generate an intraglandular communication network, suggesting its participation in the glandular activity regulation processes. The results of double-IHC might indicate the presence of IC in different functional stages, probably related to the needs of the cellular microenvironment. The morphometric variations in the proteins analyzed between immature and adult viscachas probed to be more salient in the latter, suggesting a direct relationship between the expression of the S-100 protein and GFAP, and animal age. Anat Rec, 2017. © 2017 Wiley Periodicals Inc. Anat Rec, 300:1847-1857, 2017. © 2017 Wiley Periodicals, Inc.