Natural killer cell (NK) activity in peripheral blood (PB) was followed longitudinally for up to 2 yr after initiation of low-dose IFN-α-2b therapy in nine hairy cell leukemia (HCL) patients. A whole blood NK (WB-NK) assay was employed in order to measure the NK activity per unit blood. The pretreatment WB-NK activity was consistently low, indicating that the patients' total NK activity in PB is decreased. Striking differences in WB-NK activity were observed between splenectomized and non-splenectomized patients, whereas no consistent patterns were found when using the conventional NK assay. Thus the WB-NK activity of splenectomized patients showed an immediate increase after initiation of treatment, while the activity in non-splenectomized patients decreased and remained low during the first 3-6 months. Subsequently, after reduction in spleen size, the WB-NK activity began to increase. In splenectomized patients, a second rise in WB-NK was observed after 3-6 months of therapy, coinciding with the normalisation of the peripheral blood counts. In both groups of patients incubation with IFN in vitro induced a rise in NK activity before start of treatment, which was abrogated promptly after initiation of therapy, indicating a maximal in vivo boosting of the NK cells. These differences observed indicate that the response of splenectomized and nonsplenectomized HCL patients to IFN treatment should be evaluated separately.