An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis is described. For this study expression of MHC locus II coded Ia antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein was analyzed using monoclonal antibodies in avidin-biotin-peroxidase complex staining combined with visualization of [3H]thymidine incorporation by autoradiography. Compared to spontaneous [3H]thymidine incorporation assay information is obtained at single cell level. In contrast to blast indexes calculated from MGG stained preparates, information on the expression of various functional cell surface structures as well as DNA synthesis is also obtained. Double-assay for lymphocyte phenotype and DNA synthesis by flow cytometry might be preferred to light microscopy, but the widespread use of immunoperoxidase staining and autoradiography may make this new kind of approach more easily available. Other advantages worth considering are the possibility of transporting, staining and counterstaining as microscope slides and the permanent nature of the documentation and morphological information obtained. In our experience, this method seems to be useful for studying resting peripheral blood lymphocytes as well as mitogen and antigen induced changes in the lymphocyte activation state.