Multiple methods have been used to measure antibodies to polyomavirus virions. In order to have a common method for all polyomaviruses, we developed enzyme immunoassays (EIAs) using virus-like-particles (VLPs) produced in the baculovirus expression system. We tested serum samples from humans and rhesus macaques in VLP-based EIAs for the two human polyomaviruses, BK and JC virus, and two nonhuman primate polyomaviruses, simian virus 40 (SV40) and lymphotropic polyomavirus (LPV). Rhesus sera exhibited low level reactivity to BK and JC, and approximately 10 and 15% of human sera showed low level reactivity to SV40 and LPV, respectively. Competitive inhibition assays with VLP protein demonstrated that the reactivity of rhesus sera against BK and JC VLPs was blocked by both SV40 and the respective human polyomavirus, indicating that the BK and JC assays were detected cross-reacting antibodies Similarly, the reactivity of the majority of human sera to SV40 was blocked by both SV40 and BK or JC, demonstrating that the SV40 reactivity of human sera is largely due to cross reacting BK and JC antibodies. In contrast, the reactivity of human sera to LPV VLPs was blocked by LPV but not by BK or JC, providing serological evidence for an unknown human polyomavirus related to LPV. SV40 and LPV VLP-based EIAs and competitive inhibition assays with heterologous VLPs provide tools for seroepidemiological studies of possible SV40 and LPV-like infections of humans.