A biofilm-negative transposon mutant was created from an Enterococcus faecalis strain that produces a lot of biofilm. The transposon had been inserted in the second gene of a locus consisting of 4 open-reading frames, designated bop (biofilm on plastic surfaces). A nonpolar deletion of this gene and of parts of the 2 flanking genes was created; production of biofilm by this deletion mutant was significantly enhanced, compared with that by the wild-type strain. Expression of a downstream gene was significantly lower in the transposon mutant than in the wild-type strain and the biofilm-enhanced deletion mutant. Transformation of this gene into the transposon mutant partially restored production of biofilm. Mice challenged by intravenous injection with the biofilm-negative mutant strain showed significantly reduced numbers of colony-forming units in the blood, compared with mice challenged with the biofilm-enhanced deletion mutant and the wild-type. These results indicate that bop is involved in production of biofilm and probably regulates expression of biofilm in the E. faecalis strain tested.