Abstract Oncolytic virotherapy with measles vaccine virus (MeV) already has been demonstrated to be safe. However, early clinical results pointed out the necessity for an enhancement of oncolytic effectiveness of MeV-based virotherapeutics. In our work, we are developing an armed measles vaccine virus (MeV-SCD) encoding a suicide fusion gene of yeast cytosine deaminase/uracil phosphoribosyltransferase, converting the nontoxic prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic drug 5-fluorouracil (5-FU). To preclinically investigate what an optimal prodrug-assisted therapeutic regimen might look like, we added 5-FC at various time points after infection with MeV-SCD and either let the prodrug remain in the tumor cell culture medium continuously for various time periods ("continuous" 5-FC application) or applied it only temporarily for defined shorter periods of time ("pulsed" 5-FC application); we also varied the time point at which 5-FC was added after infection with MeV-SCD. As a result, addition of the prodrug at early times postinfection (e.g., at 3 hr postinfection) was found to be inferior concerning the overall oncolytic effectiveness when compared with addition of 5-FC at later time points (e.g., at 24 hr postinfection). Next, oncolytic effectiveness was found to correlate positively with the overall duration of incubation of MeV-infected tumor cells with 5-FC. Of note, this was true despite our finding that addition of the prodrug could also exert an inhibitory effect on the generation of infectious progeny viral particles, that is, on virus replication. These findings should be helpful for the rational design of further trials (preclinical, clinical) using suicide gene armed virotherapeutics, such as MeV-SCD.