The ability of spleen cells treated with methionine enkephalin (Met-ENK) in the presence of 3'-azido-3'-deoxythymidine (AZT) to produce cytokines and inhibit Friend leukemia virus (FLV) replication in Mus dunni cell cultures was investigated. In the presence of murine spleen cells, combination treatments using AZT plus Met-ENK or concanavalin A reduced FLV replication by 63% and 84%, respectively, as compared with 47% for AZT alone. When interleukin (IL)-2, IL-4 and interferon (IFN gamma) levels were measured in FLV-infected cell cultures, both AZT and Met-ENK treatments induced a higher production of IFN gamma and a slight increase in IL-2 and IL-4, as compared with either treatment alone. Subsequent treatment of FLV-infected cells with concanavalin A-stimulated cell supernatants, containing approximately 10 U/ml each of IFN gamma and IL-2, resulted in inhibition of viral replication. Thus, in the absence of spleen cells, IFN gamma was added to cell cultures to determine whether this cytokine contributed to combination antiviral effects. Results show that addition of IFN gamma alone results in a slight suppression of FLV expression, whereas treatment with both AZT and IFN gamma inhibits FLV replication significantly. Subsequently, addition of anti-IFN gamma antibody to cell cultures treated with Met-ENK blocked antiviral effects due to this neuropeptide. Thus anti-FLV effects of spleen cells treated with Met-ENK in combination with AZT are mediated to a large degree by IFN gamma.