To investigate the adjuvant capacity of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN-gamma), we cloned these rhesus cytokines into a mammalian expression vector. Two groups of six rhesus macaques (Macaca mulatta) received intradermal immunizations of plasmid DNA coding for SIV Eng and Gag, and influenza virus nucleoprotein (Flu-NP), with or without the co-administration of plasmid DNA coding for these cytokines. Humoral immune responses to antigens of both of these viruses and SIV specific T cell proliferative responses were significantly enhanced by co-immunization with the cytokines. These twelve monkeys, and a group of six naive controls, were challenged by the oral mucosal route with the uncloned and highly pathogenic SIVmac251. All monkeys became infected. The early CD4 decline was reduced in the group co-immunized with cytokine and viral plasmids. Unexpectedly, plasma viremia set points were not different in this co-immunized group and the non-immunized control group. On the other hand, monkeys vaccinated with equivalent amounts of empty vector plasmid (i.e. no cytokine inserts) along with plasmids expressing viral antigens demonstrated a slight but significant decrease in acute viremia compared to non-immunized controls (P<0.02). However, viral loads at set points were not significantly different between both the immunized and the non-immunized control group. Thus, although the cytokine vectors demonstrated detectable enhancement of the immune response to different viral antigens, such enhanced response did not translate into better anti-viral control in our experiment. These results underscore the need for further testing of cytokines as vaccine adjuvants in relevant animal models.