The presence of hemolysin, gelatinase, and aggregation substance (by use of a probe known to hybridize to most pheromone-responsive plasmids) was determined in 192 isolates of Enterococcus faecalis from patients with endocarditis or other infections and fecal isolates from hospitalized patients or healthy volunteers, and in 86 non-E. faecalis isolates. Hemolysin was more common in nonendocarditis clinical isolates and in hospital fecal isolates (37% and 31%, respectively) than among endocarditis and community fecal isolates (16% and 20%, respectively). Gelatinase and aggregation substance, respectively, were found in 54% and 52% of isolates from endocarditis, in 58% and 72% of isolates from other infections, in 62% and 56% of hospital fecal isolates, and in 27% and 30% of fecal isolates from healthy volunteers. All 86 non-E. faecalis enterococcal isolates were negative for these traits. The absence of hemolysin, gelatinase, or the aggregation substance gene in > 45% of endocarditis E. faecalis isolates suggests that while these traits may play a role in virulence, other properties are also important.