Tissue culture growth medium derives a substantial fraction of its growth-stimulating activity from the fetal bovine serum (FBS) commonly used as a supplement. In addition to the source of serum, non-essential additives such as colorized pH indicator dyes may also affect the growth stimulating properties of complete media. We show here that both of these culture medium components can dramatically affect gene expression in vitro. Using a custom gene expression chip for herpes simplex virus 1, we demonstrated significant changes of expression levels in several categories of viral genes including the immediate early viral transcription factors ICP0 (p < 0.05), ICP4 and ICP27 (both p < 0.001). This dependence of virus growth on serum source and other medium components have implications for not only in vitro virus studies, but also viral vector design and vaccine efficacy. This is especially true when examining a large DNA pathogen that potentially contains response elements that are common in the mammalian genome...