Spatial associations of individual plasma membrane components have been proposed as being important in the functional coupling of these molecules. CD45 and Thy-1 associate on the membranes of both transformed and normal lymphocytes as measured by several different methods, and both of these molecules have been found to contribute to T cell activation and proliferation. Thy-1, however, lacks both transmembrane and cytoplasmic domains (it is linked to the plasma membrane through a glucosyl-phosphatidylinositol linkage), obliging the activation and proliferation signals that act through Thy-1 to be transduced by neighboring molecules. In the experiments reported here, the association of Thy-1 and CD45 was examined on lymphocytes from two mutant mouse strains, C3H-gld/gld and C3H-lpr/lpr, both of which exhibit lymphocyte activation and proliferation parameters significantly different from the control C3H/HeJ strain. No significant differences in Thy-1/CD45 association (measured by a heterologous epitope blocking assay) to distinguish mutant from wild-type thymocytes were found. However, spatial associations of CD45 and Thy-1 were altered on both mutant lymph node cells and splenic lymphocytes. These abnormal associations suggested possible effects on the membrane protein tyrosine phosphatase (PTPase) activity associated with CD45. Both C3H-gld/gld and C3H-lpr/lpr lymph node cells were found to have significantly elevated levels of membrane PTPase activity, and this elevation correlated with the anergy to mitogens that develops as these animals age. Finally, monoclonal anti-Thy-1 antibody (G7) reduced the PTPase activity of wild-type membrane isolates, suggesting that Thy-1/PTPase interactions may be a mechanism by which G7 provokes a lymphoproliferative response.