: Xenotransplantation may be associated with the risk of transmission of microorganisms. In particular, the porcine endogenous retroviruses (PERV) have raised concerns as in vitro experiments show susceptibility of human cells for PERV infection. However, it remains unclear whether PERVs are able to infect transplant recipients in vivo and whether they are pathogenic. It is therefore essential that the risks are evaluated and for this purpose specific and sensitive screening methods for PERVs have to be developed. We generated specific antibodies against all major structural proteins of PERV and developed several assays which allow antibodies against PERV to be detected as indirect evidence of infection. For direct detection of PERV production, reverse transcriptase (RT) assays were used. PCR methods were used to detect provirus integration and the presence of viral mRNA. Here we present an immunoperoxidase assay (IPA), which would allow the detection of viral proteins in infected cells as well as antibodies against PERV in the serum of an infected host. The specificity of the sera used in the assay was determined by several methods, including immunoelectron microscopy, and the sensitivity of the assay was compared with other methods. This IPA was used to detect PERV infection in in vitro experiments for evaluation of the virus host range, for titrating the virus and for testing anti‐viral properties of AZT. Using this method it was shown that AZT inhibits replication of PERV. This IPA may be very useful for the surveillance of preclinical and clinical xenotransplantations.