We have developed a rapid and efficient nucleotide sequencing technique, named the colony direct sequencing method, which combines both the conventional cloning method for picking up a single gene and the polymerase chain reaction (PCR) method for amplifying the gene directly from a colony. In the present study, the colony direct PCR product was used both for identification of the DNA insert and for nucleotide sequencing by an automated DNA analysis system. A nucleotide sequence of 300 to 400 bp could be determined within 13 h after picking the bacterial colonies on LB medium plates. We applied this method to sequencing of junctional regions of multiple deleted mtDNAs in two siblings with inherited recurrent myoglobinuria. Mitochondrial DNA fragments with deletions were amplified by PCR and then cloned into plasmids. Among 48 white colonies propagated on LB medium plates, nine different clones were identified by PCR directly from colonies. Determination of six different junctional sequences disclosed involvement of directly repeated sequences of 2 to 12 bp in length on each side of the deletions. We believe that the colony direct sequencing method will be a powerful tool in molecular genetics for identification of a single gene among polymorphic DNAs.