Chromosomal size polymorphism in Leishmania of subgenus Viannia has been correlated with eco-geography. The sizes of chromosomes bearing rDNA genes were determined in 69 isolates. A considerable size-variation was observed, ranging from 1100 to 1500 kb. Chromosomes of L.(V.). braziliensis, L.(V.)guyanensis and L.(V.) peruviana from northern Peru were significantly larger (200 kb) than those of L.(V.) peruviana from southern Peru. In addition, 31 out of 69 isolates presented each two different-sized homologues of the rDNA chromosome. Long range restriction mapping of three different-sized rDNA chromosomes from L.(V.)braziliensis M2903 and L.(V.)peruviana HB31 (north) and LC106 (south) each revealed three fragments delimited by PmeI restriction sites: two constant in size (the centre and one extremity of the chromosome) and one variable (the other extremity, containing a single cluster of rDNA genes). Further analysis of the M2903 rDNA chromosome allowed the localization of its 140 kb rDNA cluster at 85 kb from the telomeric end. Two arguments indicated that size-variation of the rDNA chromosome is partially due to amplification/deletion of the clustered rDNA genes: (i) size-variation of the cluster-containing fragment was proportional to the size-variation of the whole chromosome, and (ii) hybridization signal intensity of the rDNA chromosome with a small subunit rDNA probe strongly correlated with chromosomal size. Nevertheless, DNA sequences present between the rDNA cluster and the telomere might also play a role in chromosomal size polymorphism. In addition, our data suggest that rDNA gene copy number (20-40 copies cell(-1) under a diploid hypothesis) in subgenus Viannia is lower than reported previously.