Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in Epstein-Barr virus (EBV)-induced B lymphoblastoid cell lines (LCL) derived from bone marrow cells and peripheral lymphocytes of four patients with congenital agammaglobulinemia (CAG). In control subjects there was no appreciable difference in the range of ecto-5'-NT activity between immunoglobulin (Ig)-producing B LCL and their peripheral lymphocytes. Though peripheral lymphocytes of the patients showed lower ecto-5'-NT activities than those of control subjects, ecto-5'-NT activities in Ig-producing B LCL from peripheral lymphocytes of the patients fell into the same range as those in B LCL from control subjects. Ecto-5'-NT activities in non-Ig producing precursor B cell lines derived from bone marrow cells of the patients also registered within the range of the enzyme activity of Ig-producing B LCL from control subjects. B LCL from mothers of the patients, who were presumed to be heterozygotes, also had the same range of ecto-5'-NT activity as the control subjects. These results indicate that ecto-5'-NT does not appear to be involved in the pathogenesis of congenital agammaglobulinemia.