In the present study, the risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of oocytes with MMV-exposed spermatozoa and to resulting pups was evaluated. Also, the time of seroconversion of recipients and pups was investigated. To achieve this goal, IVF of oocytes with cryopreserved spermatozoa from the inbred C3HeB/FeJ mouse strain was performed, and the resulting embryos were transferred to suitable Swiss recipients. Three groups were investigated: 1) oocytes or the developing embryos were continuously exposed to 10⁴ TCID₅₀ MMVp per milliliter in the fertilization (human tubal fluid [HTF]), culture (KSOM), and embryo transfer (M2) media (positive control); 2) oocytes and spermatozoa were exposed to MMVp in the HTF medium only and transferred after a standard washing procedure with 10 washing steps in virus-free KSOM and M2; and 3) oocytes and spermatozoa were exposed to virus-free HTF, KSOM, and M2 (negative control). To detect antibodies to MMV in recipients and progeny, serological analyses were performed by ELISA on Days 14, 21, 28, and 42, and on Days 42 and 63, respectively, after embryo transfer. The presence of MMV in the washing drops was analyzed by PCR and an in vitro infectivity assay, while organs of some recipients and pups were analyzed by PCR. Using 10⁴ of the tissue culture infective dose of MMVp per millilitre in the fertilization medium only, the present results demonstrate that 10 washing steps in the IVF-ET procedure are sufficient to remove the virus to a noninfectious dose, producing MMV-free seronegative recipients and pups. As such, there is minimal risk of transmission of MMV to recipients and pups if spermatozoa become contaminated with such viral loads.