We developed a rapid extraction method to analyze the chromosomal DNA of 84 isolates of Campylobacter pylori (formerly Campylobacter pyloridis) from 70 individuals. Only three of the nine endonucleases tested gave satisfactory digestions: HindIII, EcoRI, and SacI. The latter two produced mostly larger bands, whereas HindIII produced smaller bands, which allowed clearer comparisons between isolates. The isolates from 69 Australian subjects and one from England each had a unique profile. Isolates from a husband and wife were different, as were those from a brother and sister. In pairs of isolates from 11 individuals, the second isolate was markedly different in six subjects. The profile changes were not associated with changes in antibiotic susceptibility or with loss of catalase or urease activity, which can occur during storage of C. pylori. These restriction endonuclease profiles suggest considerable subspecies variation in C. pylori. Plasmid bands were found in undigested DNA from 40 of the 84 C. pylori isolates.