In order to evaluate whether bovine herpesvirus-1 (BHV-1) could be used as a live viral vector for the expression of cytokines, we constructed a recombinant BHV-1 expressing bovine interleukin-1 beta (boIL-1 beta). The boIL-1 beta coding sequence, corresponding to the cleaved mature product, was fused with the BHV-1 glycoprotein C (gC) signal peptide sequence; the resultant gC-boIL-1 beta fusion gene was recombined into the gC locus of the BHV-1 genome. Southern blot analysis confirmed the proper genomic configuration of the recombinant virus. Results from transcript analysis showed that boIL-1 beta was expressed in infected cells with kinetics similar to that of gC. Indirect immunofluorescence and immunoprecipitation assays showed that the recombinant protein was produced in both cell-associated and secreted forms. Western blot analysis detected a 19.3-kDa protein. Further analysis, using an IL-1 beta bioassay demonstrated that both the cellular and secreted forms of recombinant boIL-1 beta possessed biological activity. The expression of the boIL-1 beta protein did not affect the in vitro growth efficiency of the virus, which exhibited similar growth kinetics to that of a simple gC deletion mutant. The results from this study demonstrate that BHV-1 can be used to express a functional cytokine, thereby establishing the basis to further study recombinant BHV-1 expressing cytokines as an alternative means to attenuate the virus and also as a potential in situ cytokine delivery system to modulate immune responses against BHV-1 and other cattle pathogens.