Interferon (IFN)-alpha has a therapeutic effect in several B cell malignancies, including low-grade non-Hodgkin's lymphoma (NHL), multiple myeloma, and hairy cell leukemia, whereas its efficacy in the treatment of B cell chronic lymphocytic leukemia (B-CLL) is rather limited. In the present study, we investigated the effect of IFN-alpha on the biologic functions of B-CLL cells, which were stimulated by cross-linking of the CD40 antigen. In cell samples from 16 B-CLL patients, the addition of IFN-alpha to CD40-stimulated purified B-CLL cells caused a significant increase in [3H]thymidine uptake (p < 0.003). In B-CLL cells maximally activated by CD40 cross-linking and interleukin-2 (IL-2)/IL-10, proliferation was not further enhanced or inhibited by IFN-alpha. In contrast, proliferation of normal tonsillar B cells stimulated by the combination CD40/IL-2/IL-10 was significantly inhibited by IFN-alpha. Because B-CLL activation might be enhanced by induction of the autocrine growth factor tumor necrosis factor-alpha (TNF-alpha), we investigated the effect of IFN-alpha on the secretion of B cell tropic cytokines. In fact, IFN-alpha significantly stimulated the secretion of IL-6 and TNF-alpha in CD40-activated B-CLL cells (p < 0.01). Although exogenous addition of TNF-alpha did not influence activation of CD40-stimulated B-CLL cells, neutralization of TNF-alpha by polyclonal antibodies led to a complete inhibition of CD40-mediated proliferation of B-CLL cells, suggesting that enhanced proliferation and increased cytokine production by CD40-activated B-CLL cells are independent IFN-alpha-mediated events. The studies presented here provide evidence that IFN-alpha mediates costimulatory signals in the context of T cell-mediated B-CLL cell activation.