Part of the human mitochondrial D-loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M13-21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single-stranded DNA was generated. This was then sequenced directly by the diodeoxy chain termination method using dye-labelled universal sequencing primers in conjunction with a fluorescence-based DNA sequencer. This enabled a 403-base-pair hypervariable segment of the D-loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.