An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutations by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost the entire mitochondrial genome, which then serves as template for the amplification of 25 short PCR fragments in two multiplex groups corresponding to regions implicated in human diseases. The mixture of fragments was subsequently subjected to two-dimensional electrophoretic separation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamide gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differences in melting behavior of mutant and wildtype fragments. Evaluation of the method using samples with known homoplasmic and heteroplasmic mutations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously identified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In addition, two variants were found to be novel mutations when analyzed by sequence analysis. One of these novel mutations was a heteroplasmic mutation in the COXIII gene that was found to segregate to homoplasmy in the next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected.