Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[3H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC50 (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population. © 2002 Elsevier Science (USA).