The expression of Ia antigens on murine thymocytes has been assessed by indirect immunofluorescence and flow microfluorometry analysis. Antisera specific for I-region determinants were found to react with at least 50% of thymus cells in single-cell suspensions. When these antisera were tested on thymus cells of an appropriate recombinant strain, it was found that both I-A and I-E/C subregion determinants could be detected. Monoclonal antibodies to I-A and to I-E determinants also reacted with thymus cells in a similar fashion, eliminating the possibility that contaminating specificities accounted for the reactivity of alloantisera. The majority of Ia-positive thymus cells appeared to express determinants encoded by genes of both I-A and I-E/C subregions. Ia expression on individual thymocytes was also correlated with cell size as measured by forward light scatter intensity. However, unlike antigens detected by rabbit anti-mouse brain antisera, Ia antigens did not appear to be associated with known subpopulations of thymocytes distinguishable by light scatter intensity. The presence of I-region encoded antigens on thymocytes may play a role in maturation of T cells in the thymus microenvironment.