Trypanosomatids are characterized by the presence of kinetoplast DNA (kDNA), a peculiar form of mitochondrial DNA that consists of several thousand minicircles and a few dozen maxicircles catenated in a network. Within a species, the minicircles are known to differ in nucleotide sequence, but are homogeneous in size and always cross-hybridize. In all species of trypanosomatids, kDNA minicircles have at least one copy of a conserved 100-200 nucleotide region containing an almost invariant 'universal' 12-mer sequence (5'-GGGGTTGGTGTA-3'). We here report that Trypanosoma rangeli, a non-pathogenic parasite of man, contains two distinct classes of kDNA, minicircles called KP1 and KP2, which differ in size and molecular organization. Both were cloned and sequenced in both directions. KP2 was 1587 bases along and contained two copies of the conserved region as direct repeats 180 degrees apart. In contrast, KP1 had 1764 bases and showed a single conserved region. Moreover, KP1 differed further from KP2 and from most other previously sequenced trypanosomatid minicircles by containing a nucleotide substitution (5'-GGGGTTAGTGTA-3') in the 12-mer universal sequence tag. Polymerase chain reaction and hybridization studies suggest that the sequence of KP1 is very conserved in several other T. rangeli strains from Honduras, Colombia and Venezuela. It thus could provide a good target for the molecular diagnosis of infection with this parasite.