We have used a new protocol of prolonged in vivo hydroxyurea (HU) administration which eliminates all cycling and short-lived cells. This treatment kills 99% of non-B non-T bone marrow cells, and it leaves in spleen and bone marrow "long-lived" B- and T-cell populations which represent 33 and 59%, respectively, of the total numbers of lymphocytes found in untreated controls. The relative proportions of B and T cells in spleen or blood of HU-treated mice were practically unaffected, while an increased blood-to-marrow permeability results in markedly abnormal proportions of B and T lymphocytes in bone marrow. Mitogen reactivities of these long-lived lymphocytes recovered either in spleen or bone marrow of HU-treated animals were studied. The results show that such B cells respond perfectly well to the B-cell mitogen lipopolysaccharide, by proliferation and differentiation into Ig-secreting cells, and that T cells proliferate at nearly control levels in response to concanavalin A. This protocol of long-term HU treatment offers the possibility of studying selected long-lived lymphocyte populations, the clonal repertoires and functional properties of which can now be readily approached.