Dipeptidyl peptidase IV (DP IV, CD26), known as an activation marker of T lymphocytes, is a proline-specific protease thought to be involved in the regulation of the immune response. The physiological role of dipeptidyl peptidase IV in the immune system and the molecular events of lymphocyte activation mediated by this enzyme are only partly established. Former results suggested the occurrence of different molecular forms of DP IV in distinct human sources. As yet it has been unknown whether DP IV from human hematopoietic cells also appears in different forms and whether similar structural modifications are involved in functional processes of the regulation of the immune response. Here we describe that lymphocytic DP IV/CD26 occurs in various molecular forms and that some of them are associated with the activation process. In cell lysates of mitogen-activated lymphocytes at least 5 enzymatically active DP IV forms and up to 11 immunoreactive molecular forms of this enzyme with isoelectric points between pH 3.5 and 5.9 were discernible. Corresponding analyses of soluble and membrane cell fractions of human lymphocytes showed significant differences in the staining pattern of molecular DP IV structures. After mitogenic stimulation a special molecular form of DP IV arises in the membrane, which was originated either from the soluble part of the cell (translocation) or represents a new synthesized form. Particularly, changes of molecular DP IV forms after mitogenic stimulation strongly suggest that special forms/epitopes of this enzyme are directly involved in the process of lymphocyte activation and growth. Importantly, different monoclonal DP IV antibodies partly define different molecular forms of DP IV. Moreover, the pattern of immunostaining and enzymatic staining (Gly-Pro-beta-methoxynaphthylamide) also reveals drastic differences. These data strongly suggest a direct relationship between the expression/recognition of special DP IV epitopes and the contradictory functional effects of monoclonal DP IV antibodies found by us and other groups.