In the mouse model, the arbovirus Venezuelan equine encephalitis virus (VEE) replicates in lymphoid tissues prior to either inducing protective immunity (attenuated VEE mutant) or progressing to lethal encephalitis (virulent parent VEE). To investigate the mechanism of the protective response, cytokine gene expression was examined during the course of the primary in vivo immune response to molecularly cloned, virulent VEE and a single-site attenuated VEE mutant, using a quantitative reverse transcriptase-polymerase chain reaction assay. VEE-induced cytokine gene expression was 100-fold elevated over that of untreated controls for IFN-gamma and IL-6 and 10-fold increased for IL-12, IL-10, and TNF-alpha. There was no qualitative difference in cytokine gene induction comparing mice infected with the attenuated and the virulent VEE; however, there were significant differences in the cytokine gene expression kinetics. In mice infected with the attenuated VEE, elevated cytokine gene expression was delayed 24 hr when compared to mice infected with the virulent parent VEE clone at the same dose. Further, IFN-gamma protein secretion by cells from the draining lymph node mimicked the pattern of IFN-gamma gene induction by cells harvested from the same site. IFN-gamma gene expression was elevated at an earlier time point in mice given virulent V3000 24 hr after attenuated V3032 injection compared to mice infected with virulent V3000 alone. The combined V3000/V3032 infection resulted in host protection. Treatment of mice with IL-12 prior to infection with virulent VEE failed to reduce the severity of infection, while anti-IL-12 antibody did not prevent the early protective effect of attenuated virus. In contrast, administration of anti-IFN-alpha/beta antibody prior to VEE infection worsened virulent VEE disease. These results indicate that the attenuated VEE strain elicits a similar but delayed cytokine response compared to the virulent strain, suggesting that the kinetics of cytokine expression and the particular cytokine produced may influence the development of a host protective response. Furthermore, IFN-alpha/beta, but not IL-12, seems to be a major factor in the induction of early protection against VEE infection and disease.