Murine parvoviruses, including minute virus of mice (MVM), represent major infectious disease problems encountered in contemporary laboratory animal research facilities with embryo transfer (ET), one of the most widely used techniques for rederivation. Using an in vivo approach, the objectives of this study were to assess the risk of MVM transmission during rederivation and to provide data that allow recommendation of preventive measures. Therefore, we determined whether immunosuppressive variant MVMi viral DNA is detectable in reproductive organs, gametes (oocytes and spermatozoa), and embryos collected from experimentally infected mice and whether washing as recommended before ET eliminates MVMi sufficiently from gametes and embryos. Fractions of reproductive organs tested positive from Day 5 to Day 30 postinoculation, demonstrating a risk for a minimum period of 4 wk; the highest incidence of positive organs was found between Day 9 and Day 13 postinoculation. Real-time PCR detected viral DNA to a lesser extent in male than in female reproductive organs. MVMi DNA was detected in oocytes and sperm cells derived after in vivo infection but not in two-cell embryos. In vitro contamination studies revealed that the virus firmly adheres to the zona pellucida after 10 wash steps, indicating that even extensive washing might not eliminate MVMi completely from embryos. According to this systematic in vivo approach, recommended measures to prevent transmission of MVM during rederivation include sufficient washing of embryos, accompanying testing using adequate (PCR) methods, and using embryos rather than in vitro fertilization techniques; furthermore, the exchange of gametes should be considered a risk factor.