Active screening for vancomycin-resistant enterococci (VRE) in rectal and stool specimens has been recommended to limit the spread of antimicrobial resistance within certain high-risk populations. Directly from 502 rectal swabs and stool specimens, we evaluated the diagnostic performance of the BD GeneOhm VanR assay (BD GeneOhm, San Diego, CA), a rapid real-time PCR test that detects the presence of vanA and/or vanB genes. The VanR assay was compared to culture consisting of both bile-esculin-azide agar with 6 mug/ml vancomycin (BEAV agar) (BD Diagnostics, Sparks, MD) and BEAV broth with 8 mug/ml vancomycin (Hardy Diagnostics, Santa Maria, CA). Enterococci were identified to the species level using standard biochemical tests and a Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). The susceptibility of the enterococci to vancomycin and teicoplanin was determined using an Etest (AB Biodisk, Solna, Sweden). VRE were initially isolated from 147 cultures, and the VanR assay detected 142 of the 147 positive cultures for a sensitivity of 96.6%. The specificity was 87.0% (309/355) largely due to false positives seen with the vanB portion of the assay. The sensitivity when testing rectal swabs was 98.3%, and the sensitivity for stool samples was 95.4% (P = 0.643). The specificity of rectal swabs was comparable to that of the stool specimens (87.5% and 86.5%, respectively). When used only to detect VanA resistance, the VanR assay was 94.4% (136/144) sensitive and 96.4% (345/358) specific, with positive and negative predictive values of 91.3% and 97.7%, respectively. In summary, the BD GeneOhm VanR assay is a good screening test for VRE in our population of predominantly vanA-colonized patients. However, patient samples testing only vanB positive should be confirmed by another method for the presence of VRE.