Using degenerate oligonucleotides complementary to sequences encoding conserved amino acid motifs in D-alanine-D-alanine (Ddl) ligases, we have amplified ca. 600-bp fragments from Enterococcus casseliflavus ATCC 25788 and Enterococcus flavescens CCM439. Sequence analysis of the amplification products indicated that each strain possessed two genes, ddlE. cass. and vanC-2, and ddlE. flav. and vanC-3, respectively, encoding Ddl-related enzymes. The fragments internal to the vanC genes were 98.3% identical. The vanC-2 gene was cloned into Escherichia coli and sequenced. Extensive similarity (66% nucleotide identity) was detected between this gene and vanC-1 from Enterococcus gallinarum (S. Dutka-Malen, C. Molinas, M. Arthur, and P. Courvalin, Gene 112:53-58, 1992), suggesting that the vanC genes are required for intrinsic low-level resistance to vancomycin. The partial deduced amino acid sequences of ddlE. cass. and ddlE. flav. were identical and closely related to that of the Ddl ligase of Enterococcus faecalis (79% identity). In Southern hybridization experiments, only DNA from E. casseliflavus and E. flavescens hybridized to probes internal to the vanC-2 and ddlE. cass. genes.