A simple and highly sensitive method, dot immunobinding assay, has been developed to select of monoclonal antibodies recognizing differential antigens. Antigen as low as picogram level can be detected by monoclonal antibody at concentrations even lower than 0.1 microgram/ml. This technique has successfully identified six differential antigens which are either increased (three) or decreased (three) during T cell activation. The differential properties were further confirmed by direct visualization of the resting and activated T cells with immunofluorescent staining or by indirect evidence when resting and activated T cell extracts were prepared and analysed with immunoblot assay. CC-98 (105 KD) was found in the nuclei, principally associated with the nucleoli. Activated T cells expressed a highly intense immunofluorescent pattern at their nucleolar localization compared to that of resting T cells. SH-1 antigen with a diffuse molecular weight range of 90-145 KD was expressed mainly in resting T cells and rapidly decreased, especially the high molecular weight species (120-145 KD), to a very low level in the activated T cells. SH-1 was identified as an intracellular molecule. NY-16 and CC-262 are identical with characteristics ascribed to those of the major histocompatibility class I antigen. Cell surface molecules CC-80 (135 KD) and CC-86 (135 KD) were decreased, while CC-196 was increased during T cell activation. These six molecules, with their differentiating properties, may have very important functions related to cell activation.