The use of organs from transgenic pigs for xenotransplantation may be associated with the risk of transmission of microorganisms, especially when the transgenic pigs express human proteins influencing complement activation. The porcine endogenous retroviruses (PERVs) are of particular concern as they can infect human cells in vitro. However, it is unknown whether PERVs can infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential for experimental and clinical xenotransplantation procedures that specific and sensitive screening methods for PERVs are established. We developed Western blot and enzyme-linked immunosorbant assays (ELISA) based on purified PERVs produced by pig and human cells or recombinant viral protein and synthetic peptides corresponding to PERVs' transmembrane envelope protein, respectively. PERV-specific anti-sera generated against purified virus particles, purified viral proteins and synthetic peptides served as positive controls. Both assays were used for screening the sera of healthy blood donors, pregnant women, patients treated with pig tissues, and butchers with extensive contact to living porcine material to detect antibodies against PERV. None of the individuals showed an antibody pattern characteristic for retroviral infections. Some individuals had antibodies reactive against the major capsid protein p27, against smaller viral proteins of the group specific antigen (Gag) in Western blot assays, or against peptides in the ELISA, probably due to cross-reactivity. Here, we present specific and highly sensitive screening methods applicable for future xenotransplantation procedures, but using these methods we found no evidence of PERV-infection among humans potentially at risk.