Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP-positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%--98%) possessed trace activity. Such patients have a high number of Ig-bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a "nonmembrane" marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.