Ia antigens have been found on the surface of B lymphocytes, macrophages, epidermal Langerhans cells and on certain transformed cells. Ia antigens have also been detected on the surface of thymocytes but the biosynthesis of these antigens by thymocytes has been difficult to demonstrate. We describe the labeling of murine thymocytes with 35S-methionine and the subsequent analysis of Ia antigens by two-dimensional polyacrylamide gel electrophoresis. Cell elimination experiments demonstrated that the Ia antigens detected were not of B cell origin and were synthesized by a Thy-1-positive thymocyte. Ia antigens from thymocytes were found to be indistinguishable from spleen Ia preparations. Since T cell I region determinants have been postulated to be involved in cellular recognition phenomena, models addressing this recognition must allow for the observation that T and B cell I region molecules detected by antisera such as A. TH anti-A. TL are indistinguishable by two-dimensional gel analysis and are thus unlikely to be involved in the generation of specificity in recognition.