Replication-conditional herpes simplex virus (HSV)-based vectors have great potential in the treatment of various types of cancers including brain tumors. HSV mutants lacking the U(L)39 gene and both copies of the gamma(1)34.5 gene (e.g. MGH1, G207) have been demonstrated to possess oncolytic effects as well as potent anticancer vaccination effects without compromising safety. Such mutants thus provide optimal templates to produce novel oncolytic HSV vectors for cancer gene therapy applications. In order to accomplish quick and efficient construction of oncolytic HSV vectors, a novel BAC-based method designated as 'HSVQuik system' was developed. This system sequentially utilizes two different site-specific recombination systems to introduce virtually any transgene cassettes of interest into the deleted U(L)39 locus (Flp-FRT in Escherichia coli) and to release the vector genome sequence from the procaryotic plasmid backbone (Cre-loxP in Vero cells). Taking advantage of the HSVQuik system, we constructed three oncolytic HSV vectors that express mouse IL4, CD40 ligand and 6CK, respectively. In vivo therapeutic experiments using two luciferase-labeled syngeneic mouse brain tumor models revealed that expression of these immunomodulators significantly enhanced antitumor efficacy of oncolytic HSV. The HSVQuik system, together with luciferase-labeled tumor models, should expedite the process of generating and evaluating oncolytic HSV vectors for cancer gene therapy applications.