CD73 is a glycosyl phosphatidylinositol-anchored protein with both ecto-enzyme activity (ecto-5'-nucleotidase) and signal transducing capabilities for human T lymphocytes. We now report an analysis of the distribution and function of CD73 in murine lymphoid tissues made possible by the development of the first monoclonal antibodies (mAb) specific for murine CD73. Subsets of T and B lymphocytes are CD73+ and the level of expression increases with lymphocyte maturation in both species. Among B cells, CD73 is largely restricted to cells which have undergone isotype switching. The signal transmitting function of CD73 is also conserved, as splenic T cells treated with anti-CD73 mAb plus phorbol 12-myristate 13-acetate proliferate and secrete IL-2. Fyn-/- mice are unresponsive to CD73 ligation, however, demonstrating the requirement for this tyrosine kinase in CD73-mediated signal transduction. CD73 is down-regulated after mAb plus cross-linking, suggesting that expression may be controlled by interaction with a ligand. Only small numbers of thymocytes are CD73+, so CD73 receptor functions are unlikely to be important for developing T cells. However, immunohistochemical analysis reveals that reticular and vascular cells throughout the thymus and other lymphoid tissues are markedly CD73+. Therefore, CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and/or function.