The polymorphisms of human mitochondrial DNA were studied in about 150 Sardinians from Cagliari and 100 other Italians living in Rome, using total blood cell DNA and the following restriction enzymes: HpaI, BamHI, HaeII, MspI, AvaII and HincII. 1. Seven different new morphs have been identified, one for HaeII, four for AvaII and two for HincII. 2. 16 and 17 mtDNA types were observed in the Sardinian and Roman samples, respectively. Of these only seven were shared by both groups. The morphs BamHI-3, MspI-4 and AvaII-9 were found associated at a frequency (10.0%) much higher than expected (0.17%). 3. Sardinians can be differentiated from the other Italians for a higher frequency of both morph AvaII-1 (P less than 0.05) and type 1 (2-1-1-1-1) (P approximately less than 0.03), and for a lower intragroup heterogeneity (0.52 v. 0.61). 4. The Italian sample on the whole can also be differentiated from the Caucasian group previously examined for a lower frequency of BamHI morph 2 (P Yates less than 0.01), a higher frequency of HaeII morph 1 (P Yates less than 0.02) and for the presence at a non-negligible incidence (5 individuals out of 229) of the new type 57-2 (2-3-1-4-13-2). The data indicate that mtDNA polymorphisms have not only proved to be a useful tool in detecting differences among major human groups but they can also differentiate populations within the same major ethnic division.