Nine Trypanosoma cruzi isolates not examined previously for kDNA structure were characterized by (a) endonuclease restriction analysis of mini-circles, followed by agarose gel-electrophoresis of digests, and (b) hybridization of mini- and maxi-circle fragments with four 32P-labeled cloned mini-circles from T. cruzi (pTck-1, 12, 13 and 14) or with 32P-labeled maxi-circles from T. brucei, respectively. The gel electrophoresis patterns demonstrated significant differences between isolates, which were confirmed and extended by the hybridization assay. When using pTck-1 and pTck-12 as probes, widely distributed heterogeneous mini-circle subpopulations were demonstrated in all the examined isolates, despite the occurrence of extensive homologies. pTck-14, assayed under high stringent conditions, detected an almost homogeneous mini-circle subpopulation in only three isolates, although under relaxed conditions, pTck-14 shared sequence homologies with most of the mini-circle subpopulations from all isolates. Rapidly evolving mini-circle regions were also detected using as probe pTck-13, a small mini-circle fragment. Preliminary maxi-circle characterization revealed polymorphic restriction endonuclease sites in the different T. cruzi isolates. These results were consistent with those obtained with mini-circles subjected to the same treatment.