To assess the frequency of extracellular superoxide (O-2(-)) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19443), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25788), and Enterococcus gallinarum (ATCC 35038) produced extracellular O-2(-). Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O-2(-). Mean (+/-SE) rates of extracellular O-2(-) production in vitro by E. faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4+/-0.2, 1.9+/-0.2, and 1.5+/-0.3 nmol of O-2(-) min(-1) 10(9) cfu(-1), respectively (P = .025, analysis of variance), suggesting an association between invasiveness and extracellular O-2(-) production.