Two minicircles of Trypanosoma lewisi kinetoplast DNA, linearized by different restriction enzymes, have been cloned in M13 derivative vectors. Their nucleotide sequences share a DNA fragment which accounts for 10% of the minicircle total length. This highly conserved region (constant region) is present twice within the same minicircle in a direct orientation and in an almost symmetrical location. Comparison of the T. lewisi constant region with those of T. brucei and T. equiperdum shows identical short nucleotide blocks which are shared by all the minicircles so far analyzed.