A glucocorticoid-inducible transcription unit, composed of reiterated steroid-responsive cis elements, a synthetic promoter, and the human growth hormone gene (hGH), was inserted into herpes simplex virus type 1 (HSV-1) amplicon vectors to determine if regulated expression can be achieved in cell lines and primary hepatocytes. Initial constructs were prepared in a HSV amplicon vector that contained a 0.96-kb DNA fragment encompassing an origin of DNA replication of the short repeat (ORIs) of HSV-1, similar to ORIs-containing fragments used in many of the existing HSV amplicon vectors. In the absence of glucocorticoids, these constructs produced a high basal level of hGH expression in cells both transfected with naked amplicon DNA and infected with packaged amplicon virus. To lower basal expression, the larger ORIs was replaced with a 237-bp ORIs DNA fragment that was devoid of the many trans-activation elements flanking the larger ORIs, but that could still support DNA replication of amplicon vectors. HSV amplicon vectors with this smaller ORIs produced up to 30-fold hGH induction upon glucocorticoid treatment in virally transduced 293 cells, and up to 50-fold induction in transduced primary rat hepatocytes. The kinetics of hGH accumulation after induction and the dependence of hGH expression with respect to dexamethasone concentration were characterized in both cell types. These vectors have application for experiments in which highly efficient gene transfer and inducible gene expression are required.