We investigated whether altering control of expression of an essential gene of pseudorabies virus (PRV) influences virus replication and virulence. The PRV immediate early (IE) gene was selected as a target, and its promoter was replaced with the promoter of the heat-shock gene HSP70 of the fruit fly Drosophila. The HSP70 promoter was selected because it is well characterized and can be induced in a broad range of eukaryotic cell lines at temperatures around 42 degrees C. Overlap recombination was used to construct the NIA3-HSP mutant virus. When stocks of the recombinant virus were titrated at 42 degrees C, virus titres were 100 times higher than titres obtained at 37 degrees C. Once replication began, however, the rate of growth of the mutant NIA3-HSP was equal at both temperatures. When wild-type virus was titrated at both temperatures, titres were identical. Mice that were infected with the mutant virus had a longer mean-time-to-death than those infected with the wild-type virus. Thus, the mutant virus was considered to be less virulent. We conclude that replication and virulence of PRV can be modified by altering control of expression of the viral IE gene.