We have determined the apparent and actual spontaneous mutation frequencies and rates for different species and strains of the thermoacidophilic crenarchaeote Sulfolobus. The proportion of mutations caused by insertion sequences has also been analyzed. Mutation frequencies for S. islandicus (0.08–0.6 mutations per cell division and 10⁷ cells) were below those determined for S. solfataricus and comparable to or lower than those for S. acidocaldarius. The proportion of insertion sequence mutations for the S. islandicus strains REN1H1 (9 out of 230) and HVE10/4 (0 out of 24) was found to be considerably lower than in S. solfataricus P1 and P2 and also low in comparison to other S. islandicus strains. Mutants defective in either the pyrEF genes or the lacS gene have been isolated. Their growth phenotype on selective and non-selective medium was examined and the inactivating mutations in either of the genes were determined. In addition the reversion frequencies for these mutants were measured and found to be in the range of <0.6–1.5 mutations per cell division and 10⁸ cells. However, when being subjected to electroporation as a transformation procedure, increased reversion was observed.