Two-dimensional electrophoresis was used to analyze the surface glycoproteins of murine thymocytes and lymph node cells. Two-dimensional maps of unselected, radioiodinated lymphocyte surface proteins were complex, showing at least 20 different components, but simpler patterns were obtained by using rabbit antibodies directed against the surface proteins of a T lymphoma cell line, to precipitate xenoantigens from lysates of radioiodinated or biosynthetically labeled thymocytes and lymph node cells. These xenoantibodies precipitated 12-13 distinct components from each cell type, of which all but 3 were sialoglycoproteins. Two types of difference between the surface glycoproteins of thymocytes and peripheral lymphocytes could be detected. First, higher mol. wt. glycoproteins and Thy-1 are more acidic in peripheral lymphocytes than in thymocytes, and this difference disappears after neuraminidase treatment. One additional high mol. wt. glycoprotein is also detectable in peripheral lymphocytes, probably reflecting the greater carbohydrate complexity of these molecules, when expressed on such cells. Second, 3 glycoproteins are strongly labeled only on thymocytes, and 3 others only on peripheral lymphocytes. These 6 glycoproteins might represent genuine differentiation antigens.