Abstract Molecular weight and charge microheterogeneity of Thy-1 glycoprotein expressed on different T-cell populations were compared by two-dimensional gel electrophoresis and endoglycosidase treatment. Thy-1 was immunoprecipitated from detergent lysates of radioiodinated T-cells of spleens, lymph nodes, Peyer's patches, peripheral blood, IL-2-cultured thymocytes and peanut agglutinin (PNA) separated thymocytes. In general, thymocytes and IL-2-cultured thymocytes expressed the highest levels of Thy-1 with a large M r and charge variation. The Thy-1 of these cells was basic and contained low levels of sialic acid. On the other hand, peripheral T-lymphocytes exhibited a restricted M r and more limited charge heterogeneity with higher amounts of sialic acid. The Thy-1 glycoprotein of mature, low Thy-1 PNA − thymocytes had M r and charge heterogeneity identical to peripheral T-lymphocytes. The M r and charge variation of immature PNA + thymocytes was essentially identical to that of whole thymocytes. The molecular source of M r heterogeneity in thymocyte Thy-1 and restricted M r, in lymph node Thy-1 was studied by analysis with endoglycosidase-H (Endo-H) and Endo-D. The results of Endo-H digestion showed that most of the Thy-1 polypeptides from both thymocyte and lymph node contained one high-mannose type oligosaceharide which was relatively small and not heterogeneous with respect to M r. The complex-type oligosaccharides (Endo-D-sensitive) were larger and were responsible for the broad M r-heterogeneity found in thymocyte Thy-1. Both thymocyte and lymph node Thy-1 generally appear to contan three N-linked oligosaccharides (one high-mannose and two complex) and sequential hydrolysis with Endo-H and Endo-D resulted in an unglycosylated polypeptide with an M r of approx. 12,500.