Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional Herpes simplex virus 1 (HSV-1) expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. In animal models, 12 days of 5-FC administration was superior to 6 days, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing-schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng ml⁻¹) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses.