Separation of large restriction fragments by pulsed-field gel electrophoresis is a commonly used method for epidemiological typing of Streptococcus pneumoniae and many other bacterial species. Information on the genetic changes underlying the restriction fragment polymorphisms that allow discrimination between isolates is scarce. In this study fragments adjacent to ApaI sites in a clinical isolate of S. pneumoniae were cloned and used to probe HindIII and HindIII-plus-ApaI genomic DNA digests from other isolates with very different ApaI fragment patterns. If for a given isolate the HindIII fragment detected by the probe was reduced in size on digestion with ApaI, it was deduced that the ApaI site was conserved in that isolate. The results demonstrate that of six ApaI sites in PN93/908 examined, five were retained in 11 genetically different isolates and one was retained in 2 isolates but lost in 9 others. It was concluded that point mutations at restriction sites are unlikely to account for the restriction fragment length polymorphism observed and that much of the polymorphism may be due to DNA rearrangements, possibly resulting from the insertion or deletion of mobile DNA elements.