In order to obtain T cells for adoptive immunotherapy after autologous bone marrow transplantation (ABMT) for patients with resistant hematological malignancies, a culture system was developed for growing T cells and inducing non-MHC-restricted cytotoxicity using anti-CD3 monoclonal antibody (OKT3) activation. In this investigation, we show that (1) peripheral blood lymphocytes or bone marrow mononuclear cells (BMMNC) from normal donors and cancer patients can be activated with OKT3 and grown in interleukin-2; (2) normal BMMNC activated with OKT3/IL-2 exhibited non-MHC-restricted cytotoxicity and surface markers comparable to that exhibited by normal PBL activated with OKT3/IL-2; (3) both proliferation and cytotoxic functions were IL-2-dependent; (4) PBL activated with OKT3/IL-2 after cryogenic storage grew and killed comparable to PBL activated with OKT3/IL-2 prior to cryopreservation; (5) OKT3/IL-2-activated PBL and BMMNC obtained from 5 patients with non-Hodgkin's lymphomas (NHL) and 1 patient with acute myelogenous leukemia (AML) increased cell numbers 41-75-fold in 2 weeks of culture; 5 of 6 patients with NHL or AML had PBL and BMMNC that exhibited cytotoxic activity; and (6) contaminating leukemic cells did not overgrow in OKT3/IL-2-activated cultures and could no longer be detected on cytospin specimens after 3 weeks of culture. These data show that T cells in PBL or BMMNC from ABMT candidates can be activated with OKT3/IL-2 for adoptive immunotherapy in combination with ABMT.