The correlated expression of several surface antigens (CD 1, class I HLA, CD3) was examined in human unfractionated thymocytes or selected subsets by using single or double-color flow cytometry. Prethymocyte subpopulations expressed high levels of HLA. A high proportion of cortical cells expressed low levels of either HLA or CD 3 antigens. Most of these HLA+ cortical cells corresponded to the more immature cells and did not express HLA-B loci products. CD 1 a and CD 3 antigens were expressed in a high percentage of cells and the levels of expression of each antigen in individual cells were inversely correlated. These data and the contour of double-color histograms are suggestive of the existence of a single pathway of thymocyte differentiation in which class I HLA expression is switched off around the time of the initiation of CD 3 (and Ti?) expression. We suggest that the anti-major histocompatibility complex (MHC) specificity of the unselected Ti receptor may be incompatible with the expression of MHC products on the cell membrane. At this stage, CD 1 antigens, whose expression is inversely correlated with that of HLA, may fulfill the role of MHC antigens. The latter can be re-expressed later on, once the anti-MHC specificities of the Ti receptors have been selected against. Studies on in vitro modulation of HLA molecules by interferon-alpha did not reveal any correlation to the expression of CD 1 or CD 3 antigens.