Abstract Oncolytic viruses such as measles virus (MV) represent a new class of therapeutic agents that might help to overcome current limitations in cancer therapy. Although MV-based virotherapeutics already have entered clinical testing for various tumor entities, the preclinical safety of MV virotherapeutics so far has not been elucidated for particular regimens with high medical need, such as (1) direct injection into hepatic tumor sites, (2) employing high doses ibidem, and (3) concurrent usage of arming with cytotoxic genes required to further enhance oncolytic efficiency. Here, we assessed the safety and pharmacokinetics of suicide gene-armed vector MV-SCD when administered intrahepatically in two animal models, IFNAR(tm)-CD46(Ge) (interferon-α receptor deficient and CD46 MV receptor knock-in) transgenic mice and rhesus macaques (Macaca mulatta). Clinically, singular direct intrahepatic applications of MV-SCD were found to be well tolerated. Quantitative RT-PCR demonstrated the transient presence of viral RNA in various organs, whereas no shedding of infectious virus particles was observed at any time point. Histological analyses of organs did not exhibit adverse effects attributable to the test article. Blood parameters including liver enzymes revealed no deviations from normal. In both species an antiviral humoral immune response was mounted shortly after virus administration. Surprisingly, daily repeated systemic applications of MV-SCD under concomitant prodrug administration resulted in side effects in IFNAR(tm)-CD46(Ge) mice, but were less pronounced than in a 5-fluorouracil standard therapy control cohort. Taken together, these data indicate that "single shot" direct intrahepatic injections of MV-SCD in conjunction with systemic prodrug administration are safe and could be used in future virotherapeutic treatments of liver cancers.