One of the challenges in gene therapy is to ensure stable transgene expression at the site of disease with a high degree of accuracy and safety. In this paper, we examine both viral and cellular elements that may affect the level of transgene expression mediated by herpes simplex virus type 1 (HSV-1) adeno-associated virus (AAV) amplicon vectors. These elements include the AAV inverted terminal repeats (ITRs), the AAV Rep proteins, and the allelic status of 19q in human glioma cell lines. The latter is of particular interest because the AAV integration site (AAVS1) is located on the long arm of chromosome 19 and 30-40% of human glioblastoma tumors are reported to have loss of heterozygosity in this region of chromosome 19q. Fluorescence-activated cell-sorting analysis results indicate that inclusion of minimal or full-length AAV ITRs in HSV-1 amplicon vectors markedly increases the efficiency of transgene expression. On the other hand, insertion of the AAV rep gene decreases the level of transgene expression, apparently because of the cytotoxic effects of Rep proteins. Further, the levels of transgene expression appear to be independent of 19q allelic status or the number of endogenous AAVS1 sequences in the various glioma cell lines studied. Taken together, these data support employing AAV ITRs, in the context of HSV-1 amplicon vectors, to enhance short-term levels of transgene expression.