In the present study we describe the cell types containing immunoreactive vasoactive intestinal peptide (IR-VIP) in rat thymus, spleen, and lymph nodes. Indirect immunofluorescence staining and flow cytometry indicated that all lymphoid organs studied contained VIP-positive cells, with the spleen and lymph nodes having a higher proportion than the thymus. Vasoactive intestinal peptide was found in both lymphocytes and nonlymphoid cells, lymphocytes predominating among VIP-positive cells. Double immunofluorescent staining and flow cytometry showed that all lymphoid subpopulations identified contained variable proportions of VIP-positive lymphocytes. Immunocytochemical staining of cell suspensions for both light and electron microscopy showed the cytoplasmic localization of the IR-VIP. These findings, coupled to our previous results, are consistent with the idea that VIP may have a lymphoid origin and could be active in local immune responses.