Enterococcus faecalis, a commensal of the gastrointestinal tract and an opportunistic pathogen, has the ability to adhere to surfaces and form biofilms. It has been shown earlier that only 10 to 20% of an E. faecalis OG1RF culture expresses endocarditis- and biofilm-associated pili (Ebp), which are involved in biofilm formation. Another study revealed that E. faecalis clinical isolates, as well as OG1RF, are heterogeneous with respect to their apparent zeta potential, which was also correlated with increased ability to form biofilm. The aim of this study was to demonstrate that the heterogeneity in the presence of Ebp is correlated to that in apparent zeta potential. Heterogeneous cultures of OG1RF showed two distinct subpopulations with the most (-38 mV) and least (-26 mV) negative zeta potential. Deletion of EbpR, the activator of the ebp operon, or the structural genes ebpABC resulted in homogeneous culture with the most negative zeta potential. Conversely, overexpression of EbpR or the structural genes ebpABC resulted in homogeneous culture with the least negative zeta potential. The results show that ebp operon expression in E. faecalis, as measured by using P(ebp)-gfp promoter fusion, is the cause of heterogeneity in zeta potential and that pilus production causes the cells to behave as the least negative particle in an electric field.