We evaluated the usefulness of a semi-nested polymerase chain reaction (PCR) method for detecting D1S80 (pMCT118) locus polymorphisms of DNA extracted from old skeletal remains. The semi-nested PCR has been applied to the amplification of D1S80 nucleic acid sequences. For amplification of the locus D1S80, a pair of oligonucleotide primers have been used widely as described by Kasai et al. We have designed another set of primers for semi-nested PCR. This method resulted in D1S80-VNTR detection from low-titered DNA isolated from old skeletal remains. The first and second step PCR achieved amplification from as little as 10 ng and 10 pg of template DNA, respectively. Specificity and sensitivity of the amplification products was markedly improved by semi-nested PCR. In DNA extracted from biological samples, this method took about 5 hours to amplify the target DNA and 3 hours for electrophoretic separation. We demonstrated that this semi-nested PCR method was superior in sensitivity to conventional 1-step standard amplification for VNTR typing of the D1S80 locus.