Using an indirect fluorescent antibody technique, localization of thymosin alpha 1, thymosin beta 3 and thymosin fraction 6 was studied in human thymus glands ranging in age from newborn to 62 yr old. Thymosin alpha 1 was detected specifically in thymic epithelial cells of the medulla and in cells covering the cortical surface. Thymosin beta 3 was detected almost exclusively in cells covering the cortical surface. Epithelial cells in the medulla, which were positive for thymosin alpha 1, decreased in number progressively with age, beginning at around 13 yr of age. Epithelial cells positive for thymosin alpha 1 and covering the cortical surface were observed in thymus tissue from all ages, although the absolute number decreased. Several of the peptide components of thymosin fraction 6 were found in thymic epithelial cells as well as in macrophages in the thymus and spleen.