Epidemiologic evaluation of enterococci has been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared chromosomal restriction endonuclease digestion patterns of 27 isolates of Enterococcus faecalis from three different locations by using pulsed-field electrophoresis of large chromosomal fragments (14 to 1,000 kilobases). All but two isolates generated a clear, evaluable pattern with a single lysis and digestion, and the remaining two were visualized when a larger quantity of bacteria was used. All isolates from different locations generated different restriction patterns, as did most isolates within a single location; there was also evidence of spread of strains between individuals in each location. The ease with which this analysis can be performed, together with the clarity and polymorphism seen in the patterns, suggests that this technique will be very useful for epidemiological evaluations of nosocomial enterococcal infections.