Xenotransplantation from pig to humans may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) that are present in the genome of all pigs and that infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential to perform in vivo infection studies in animal models.To study PERV transmission in rats, rat primary cells and cell lines were treated in vitro with virus from different sources. Based on the assumption that susceptible cell lineages not yet tested in vitro could be present in the animal, PERV was inoculated into naïve and immunosuppressed animals. To investigate PERV transmission in a long-term exposure experiment, sera from animals grafted with pig Langerhans islet cells were tested in a Western blot assay for antibodies against PERVs. The animals were treated with streptozotocin to induce diabetes and microencapsulated and non-microencapsulated pig islet cells were applied without immunosuppression.No productive infection of a few selected rat primary cells or cell lines was observed in vitro. PERV-specific antibodies were found in none of the animals and no integration of PERV into rat cells of different organs was observed, indicating that infection had not occurred.This report demonstrates a lack of infection of rats in vivo even during immunosuppression or long-term exposure (up to 460 days) to a functioning xenotransplant. This report also shows that rats possibly due to a low receptor concentration on their cells are not a suitable animal model to study PERV transmission in vivo.