Restriction fragment length polymorphisms (RFLPs) detected in total DNA or in rRNA genes are widely used to differentiate strains of bacteria. The changes accounting for these polymorphisms and the extent of genomic difference that they reflect are generally unknown. In this report, several methods have been used to examine the DNA differences between nine Enterococcus faecalis isolates. Restriction fragments from total DNA and from rRNA genes were compared between isolates using four and five different restriction enzymes, respectively. The proportion of polymorphic fragments detected was greater with total DNA than with rRNA gene patterns, but depended considerably on the restriction enzyme used. DNA changes underlying nine RFLPs were investigated by using the polymorphic fragments as probes to test for alteration in the position of recognition sites of other enzymes. Two polymorphisms were deduced to result from point mutation in a restriction site. Six were judged to result from DNA rearrangements, five of which involved deletion/insertion of the entire probe fragment. The results demonstrate that DNA rearrangements may be responsible for a high proportion of RFLPs used to differentiate and type strains of bacteria. While this does not limit the utility of such methods, it does preclude calculation of overall DNA sequence conservation from similarities in restriction pattern between isolates. DNA sequence determination of the 16S-23S rRNA intergenic spacer of three isolates revealed minimal base substitutions (less than 1%), suggesting that overall sequence divergence between the isolates may be low.