Although pituitary adenylate-cyclase-activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide with many different immunomodulatory properties, investigations of its source in lymphoid organs are scarce. The present report contributes to the knowledge on the origin and synthesis of this peptide in immune cells of the lymphoid organs and peritoneum using immunohistochemistry, immunocytochemical staining, Western blot and RT-PCR methods. Our study reveals PACAP immunoreactivity in the thymus, spleen and lymph nodes. Cytochemical results show that PACAP is present in thymocytes, lymphocytes and plasma cells from the spleen and lymph nodes. Western blot analysis showed a band corresponding to PACAP for all lymphoid organs studied. mRNA appears in both double (CD4+CD8+)- and single-positive (CD4+CD8–, CD4–CD8+) thymocytes and in T subsets and B cells from the spleen and lymph nodes. In addition, PACAP mRNA is expressed in lymphocytes, but not in macrophages from peritoneal suspensions. Our findings show that PACAP produced by lymphocytes could be added to the growing list of mediators shared by nervous, endocrine and immune systems. Moreover, PACAP could be considered as a lymphocyte-derived cytokine in the central and peripheral lymphoid organs acting on lymphocytes and stromal cells.