ABSTRACT The VanC phenotype, as found in Enterococcus gallinarum , E. casseliflavus , and E. flavescens , is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the vanC-1 gene in E. gallinarum , the vanC-2 gene in E. casseliflavus , and the vanC-3 gene in E. flavescens have been reported, although there is some disagreement as to whether E. flavescens is a legitimate enterococcal species. Previous attempts to differentiate the vanC-2 and vanC-3 genes by PCR analysis have been unsuccessful. The purpose of the present study was to detect and differentiate the three vanC determinants and examine the distribution of these genes in a collection of both typical and atypical enterococci. The 796-bp vanC-1 PCR product was amplified only from E. gallinarum isolates. As expected, due to the extensive homology in the vanC-2 and vanC-3 gene sequences, all of the E. casseliflavus and E. casseliflavus/flavescens isolates produced the 484-bp vanC-2 PCR product, although the E. gallinarum isolates were negative. Only the E. casseliflavus/flavescens isolates produced the 224-bp vanC-3 product. Using the three sets of primers, we were able to detect and distinguish the vanC-1 , vanC-2 , and vanC-3 genes from both typical and atypical enterococci strains. Antimicrobial susceptibility tests and analysis of genomic DNA by pulsed-field gel electrophoresis were also performed, but the results indicated that they were not able to distinguish among strains possessing the three vanC genotypes.