The multiplex polymerase chain reaction-allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations in 2,000 patients suspected as having mitochondrial DNA disorders. These point mutations are classified into four categories. Category I consists of primary disease-causing, heteroplasmic point mutations. Homoplasmic nucleotide substitutions that have been reported to be possibly disease associated are in Category II. Homoplasmic nucleotide substitutions that are thought to be benign polymorphism are included in category III. The novel nucleotide substitutions recently discovered in our laboratory by single strand conformation polymorphism analysis are in category IV. Frequencies of these 44 nucleotide substitutions in 2,000 patients and 262 control individuals were studied. The results indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4% of the patients suspected as having mitochondrial DNA disorders. Since the mitochondrial disorders are a group of complex, heterogeneous, and multisystemic diseases, it is often difficult to confirm clinical diagnosis without molecular studies. Thus, the multiplex PCR/ASO method is an effective approach for initial screening of mtDNA mutations in patients suspected as having mitochondrial DNA disorders.