Ecto-5'nucleotidase (5'NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu-), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5'NT (r = .86). 5'NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5'NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5'NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5'NT+ and 5'NT- cells in their expression of other B-cell antigens. We conclude that the place of 5'NT in leukemias corresponding to early stages of B-cell development has been characterized. 5'NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.