Abstract:A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto‐5′‐nucleotidase (ecto‐5′‐NT, CD73) was produced using highly purified human placental 5′‐NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5′‐NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab′): goat anti‐mouse Ig. Both antibodies can be used to purify the three major forms of placental 5′‐NT by affinity chromatography. By two‐color immunofluorescence, CD73 was found to be expressed on 19±5% of CD3+, 11±4% of CD4+, 51±14% of CD8+, 25±8% of CD28+, 15±5% of CD29+, 27±7% of CD45RA+, and 70±6% of CD19+lymphocytes. Within T cells, CD73 expression is restricted to the CD28+subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the “light‐zone.” CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non‐keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activat