Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33degreesC than at 37degreesC and is further stabilized at 4degreesC. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33degreesC produced 2-fold higher amounts of vector than infected cells incubated at 37degreesC, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33degreesC than 37degreesC. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33degreesC yielded similar levels regardless of passage number but was reduced at 37degreesC as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.