Background: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored Results: XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. Conclusions: Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus. © 2012 Curriu et al.