Objectives: To develop a serum-based assay to detect neutralizing antibodies to the xenotropic murine leukemia virus-related virus (XMRV) retrovirus and to use this assay with polymerase chain reaction and fluorescence in situ hybridization to identify patients with prostate cancer previously exposed to XMRV infection and those who carry XMRV viral sequences in their prostate. Methods: Patients who had undergone radical prostatectomy were enrolled, and biologic specimens were obtained at surgery. The patients were genotyped for the R462Q RNASEL variant using a TaqMan genotyping assay on DNA from the peripheral blood. A serum assay that detects XMRV neutralizing antibodies was developed and used to determine which patients had serologic evidence of previous infection with XMRV virus. Some of these patients were also tested for the presence of XMRV nucleotide sequences in their prostate using polymerase chain reaction and fluorescence in situ hybridization analysis. Results: At a serum dilution of 1:150, our assay detected 11 (27.5%) of 40 patients with XMRV neutralizing antibodies, including 8 (40%) of 20 with the RNASEL genotype QQ and 3 (15%) of 20 with either the RQ or RR genotype. These results were in complete concordance with 2 other assays (polymerase chain reaction and fluorescence in situ hybridization), which were designed to detect XMRV infection. Conclusions: XMRV infects some patients with prostate cancer. Neutralizing antibodies against XMRV correlated with 2 independent methods of detecting the virus in the prostate. The antibody response suggests that with clinical serologic assay development, it might be possible to screen patients for XMRV infection. The cases presented in the present report provided biologic samples that can be used for the development of a clinically relevant assay.