TheMycobacterium aviumcomplex (MAC) includes the closely related speciesM. avium,M. intracellulareandM. paratuberculosis.The insertion elements IS900, IS901, IS1245and IS1311were used as DNA probes to characterize by restriction fragment polymorphisms (RFLPs) eight reference strains, three animal isolates ofM. paratuberculosisfrom outside New Zealand and 61 selected New Zealand MAC isolates from cattle, deer, pigs, sheep and humans. IS900was found only in strains ofM. paratuberculosis.All MAC strains contained IS1311and the RFLPs associated with this insertion element dividedM. paratuberculosisstrains into the same groups as IS900RFLPs. Except forM. paratuberculosis, all MAC strains contained IS1245and the majority of those from lesions in cattle, deer and pigs also contained IS901. All animal strains containing IS901had the same RFLPs with IS901, IS1245and IS1311. In three cases, these apparently identical strains could be differentiated by restriction fragment analysis withBstEII. IS901was not present in four human isolates or in isolates from deer without lesions. These results indicate that a very closely related group of strains causes the majority of non-paratuberculosis MAC lesions in animals in New Zealand.