Biofilms are complex aggregations of microorganisms attached to a surface. The formation of biofilms might facilitate certain survival and virulence characteristics under some situations. This study tested the hypothesis that the ability of Enterococcus faecalis to form biofilms is related to the source of the strains. E. faecalis strains recovered from root canals (n = 33), the oral cavity (n = 21), and non-oral/non-endodontic sources (n = 16) were studied. Biofilms were grown in tryptic soy broth in 96-well plates for 24 hours at 37 degrees C, fixed with Bouin's fixative, and stained with 1% crystal violet. Optical density at 570 nm (OD(570)) was measured by using a microtiter plate reader. Experiments were performed in quadruplicate on three occasions and mean OD(570) readings determined for each strain. There were no statistically significant differences between groups (p = 0.066, Kruskal-Wallis). Within the root canal and oral isolates there were no significant associations between biofilm formation and the presence of the virulence determinants asa, cylA, esp, and gelE.