The activity of the ectoenzyme 5′-nucleotidase (5′N) was determined in the T lymphocyte subpopulations from patients with chronic lymphocytic leukemia (CLL). 5′N could be detected only in the T cells from patients whose B cells also had enzyme activity. The specific activity of CLL T4 cells was 0.17 +/- 0.02 micron/h/mg protein, similar to that of normal T4 cells, which was 0.13 +/- 0.08 micron/h/mg. The CLL T8 cells, however, had a significantly lower 5′N activity (0.17 +/- 0.02 micron/h/mg) than normal T8 cells (0.41 +/- 0.11 micron/h/mg) (P = .003). Normal null cells had very low activity, while much higher levels were found in the null cells of CLL patients whose B cells had activity. These findings document a difference in activity of an enzyme between the T8 cell population of patients with CLL and that of normal subjects.