We have digested non-kinetoplast DNA from various Trypanosoma strains with restriction endonucleases and analysed the fragment distribution by one-dimensional agarose gel electrophoresis. Visual inspection of ethidium-stained gels shows differences in banding pattern between DNAs from Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum and even between different T. brucei strains, but not between three different antigenic variants derived from the same T. brucei cell. By blotting the DNA on to nitrocellulose strips the restriction endonuclease recognition sites around genes available in cloned form can be analysed by molecular hybridization and we demonstrate this for the gene which codes for one of the variant surface glycoproteins of T. brucei. The use of this method for strain classification is discussed. Renaturation analysis of T. brucei non-kinetoplast DNA shows that about 68% of this is present as single-copy DNA with a complexity of 2.5 × 107 base pairs, whereas the remainder consists of intermediate repetitive and highly repetitive DNA. The latter fraction contains a DNA which is cut by AluI into fragments of 180 base pairs, bands in CsCl at 1.690 g/cm3 and contains duplex circles heterogeneous in size and circles with duplex tails.