In eight patients with previously untreated hairy cell leukemia (HCL), by using 111In-labelled platelets and megakaryocyte quantitation, the splenic platelet pooling and the platelet production rate ( P) were evaluated before and during α-2b-interferon (IFN) treatment. Both before and after 8 months of IFN therapy the spleen was shown to pool a sizeable amount of the total body platelet mass. The average splenic platelet pools, prior to and after 8 months of IFN, were 58 ± 17 and 47 ± 11%, respectively. At the time when treatment was initiated, the patients were heterogeneous as regards the spleen size, platelet kinetics, and the bone marrow morphology. Three patients had values for P below the 95th percentile for a group of healthy control subjects; following IFN therapy they displayed a substantial increase in P. In three other HCL patients, with the largest spleens, the pre-treatment P was normal, or slightly above the values seen for the control subjects. In these patients, changes in splenic platelet pool size, blood volume, and platelet mean life-span accounted for the increase in platelet count observed in response to IFN. The mean megakaryocyte number and volume per μl bone marrow increased during IFN therapy, while the mean P remained slightly reduced. It is concluded that splenic platelet pooling would explain the previously described difference in platelet counts between splenectomized and non-splenectomized patients treated with IFN.