Subpopulations of thymic stromal cells (TSC) and T lymphocytes have been antigenically defined as a basis for understanding intrathymic cellular communications which result in the mature T-cell repertoire. In the current study we present data that the markers CD4 and Thy-1, standard T lymphocyte markers in mice, are also expressed by subpopulations of freshly isolated and cultured TSC. This was shown by flow cytometric analyses and fluorescence microscopy respectively. Use of F(ab')2 fragments of anti-CD4 mAb demonstrated specific antibody binding and not that due to FcR binding. Double-labelling experiments illustrated that the subpopulation of CD4+ stromal cells includes cells bearing the MAC-1 antigen. The majority of Thy-1+ stromal cells were epithelial cells (determined by double-labelling cultured TSC with anti-cytokeratin); however, there was also a novel subpopulation of non-epithelial stromal cells present. Whilst actual synthesis of these glycoproteins has not yet been demonstrated, their existence as surface antigens on TSC to any degree could have major relevance to the understanding of intrathymic intercellular events. It also cautions that experiments on T cell lineage relationships based on in vivo injection of anti-CD4 antibodies be interpreted with care.