The proliferation and in vitro cytolytic activity of interleukin-2 (IL-2)- activated and anti-CD3+IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti-CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P < .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3+IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3+IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3+IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2+anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]- sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC- derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK- resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti- CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL- 2+anti-CD3-stimulated cultures. In the presence of phytohemagglutinin (PHA), both IL-2- and anti-CD3+IL-2-activated cultures developed lectin-dependent cellular cytotoxicity (LDCC) against LAK-resistant targets. Thus, anti- CD3+IL-2 activation of either PBMC or MMC contributes not only to significantly increased proliferation, but also generates populations of T cells with high cytolytic potential. Cryopreserved marrow may be an acceptable alternative source of anti-CD3+IL-2-stimulated cells for future immunotherapy protocols in conjunction with marrow transplantation.