Characteristics of nosocomial enterococcal blood stream infection (NEBSI) isolates obtained from patients at 41 U.S. hospitals participating in the SCOPE Program were studied. Isolates from 480 episodes of NEBSI were characterized according to species and antimicrobial susceptibility profile. Selected isolates were also identified to species and vancomycin resistance genotype using polymerase chain reaction based methods. Polymerase chain reaction genotyping and ribotyping were used as genetic markers for molecular epidemiologic typing. Enterococci were the third most common cause of nosocomial blood stream infection in this study, accounting for 11.7% of all isolates reported. Enterococcus faecalis was the most common species (59.6%), followed by E. faecium (19.4%). Species identification errors involving E. faecium, E. durans, E. avium, and E. raffinosus were observed. Vancomycin resistance was observed in 36.4% of all participating medical centers and varied from 11.1% of medical centers in the Northwest to 60.9% of medical centers in the Southwest. Vancomycin-resistant enterococci accounted for 20.6% of NEBSI in the Northeast, 11.4% in the Southeast, 11.1% in the Southwest, and 9.5% in the Northwest regions. VanA genotypes predominated in the Northeast and Southwest, whereas vanA and vanB genotypes were equally prevalent in the Northwest and Southeast. Molecular typing studies identified strains that were unique to individual hospitals as well as strains that were prevalent in several different hospitals. NEBSI with vancomycin-resistant enterococci continues to escalate among hospitalized patients in all geographic areas of the USA.