Two monoclonal antibodies, CT1 and CT2, have recently been described that recognize cell type restricted carbohydrate determinants present on the T200 glycoprotein of murine cytotoxic T lymphocytes in vitro. I have now examined the expression of the CT antigens in the developing thymus by using immunohistology to discern in situ expression, and cytofluorographic analysis of isolated fetal thymocyte populations to analyze cell subset distribution of the CT antigens. Immunohistology demonstrated that the pattern of CT antigen expression progressed developmentally with a relatively homogeneous distribution throughout the thymus during early thymus ontogeny and a distinct subcapsular pattern late in gestation. Fluorescence flow cytometry analyses showed that at all times the CT1 antigen was expressed exclusively by a subset of the Lyt-2- L3T4-, interleukin 2 receptor+ blast cell population. In contrast, the CT2 antigen was expressed on a subpopulation of small Lyt-2+ L3T4+ thymocytes as well as on some Lyt-2- L3T4- cells. Radioimmunoprecipitation indicated that the CT1 antigen was present on the T200 glycoprotein. Although forms of the T200 glycoprotein are found on cells of all hematopoietic lineages except those of erythroid descent, the CT determinants are found only on a restricted subset of T200-expressing lymphocytes. These findings suggest a fundamental role for carbohydrate moieties and for the T200 molecule in T cell development.