we have identified a novel dna sequence of 500 bp (β500-dna) on the leishmania (viannia) subgenus, located in the intergenic region of one of the loci of the β-tubulin gene family. the sequence analysis showed that this sequence has no homology to any other sequence described so far, including the β-tubulin gene. we improved a specific β500-pcr assay, which generated a pcr product of 375 bp for total genomic dna from leishmania strains belonging to the l. (viannia) subgenus. in contrast, no amplification was found when using genomic dna from species of l. (leishmania) subgenus or other organisms. under our pcr conditions, the lower detection limit was 1 fg when a purified dna clone (plgβ4), which contains one copy of the β500-dna sequence, was used. the β500-dna pcr assay confirmed the preliminary diagnosis of cutaneous leishmaniasis in clinical samples in which the montenegro skin test was positive and parasite cultures were negative. the analytical specificity and the sensitivity of the pcr assay provide a tool for epidemiological studies of the disease.