Systemic interleukin 2 (IL-2) and IL-2-activated lymphocytes have induced tumor regression in some cancer patients. The IL-2-activated cells have usually been generated by obtaining peripheral blood mononuclear cells (PBMC) from cancer patients shortly after systemic IL-2 therapy and culturing them with IL-2 in vitro. In an effort to augment the ex vivo generation of such cells preactivated in vivo, we examined the proliferative responses of PBMC from IL-2-treated cancer patients to several proliferative signals including IL-2, interleukin 4 (IL-4), and mitogenic antibodies to CD3 and CD28. Although much is known about the response of normal PBMC to these signals, the possibility was considered that the response of lymphocytes preactivated by IL-2 in vivo might differ from that of normal PBMC. Accordingly, PBMC obtained from ten normal, healthy controls and from 17 patients with advanced cancer 1 to 3 days after systemic IL-2 therapy were cultured for 4 days with IL-4 (1000 units/ml) and/or IL-2 (10 units/ml or 1000 units/ml) or with combinations of IL-4 and anti-CD3 +/- anti-CD28, and they were then tested for proliferation by [3H]thymidine incorporation. IL-4 failed to induce proliferation of normal PBMC and inhibited IL-2-induced proliferation, whereas IL-4 alone induced proliferation in PBMC from five of 11 IL-2-treated patients and did not inhibit but augmented the proliferation induced by IL-2 (10 units/ml and 1000 units/ml) in PBMC from six of nine patients and five of 11 patients, respectively. Anti-CD3 induced proliferation in PBMC from eight of nine patients, and the proliferation was consistently augmented by coculture with anti-CD28. Finally, IL-4 significantly augmented the proliferative responses of PBMC from IL-2-treated patients to anti-CD3, as well as to the combination of anti-CD3 and anti-CD28. Thus, in PBMC from IL-2-treated cancer patients, IL-4 enhanced the in vitro proliferation induced by IL-2 or by anti-CD3 +/- anti-CD28. The results suggest that IL-4 and/or mitogenic antibodies may be useful in augmenting the ex vivo generation of lymphocytes for clinical adoptive immunotherapy.