Kinetoplast DNA (kDNA) sequence organization, transcription, and alterations in dyskinetoplastic (Dk) mutants were examined in Trypanosoma brucei, using physically isolated and recombinant kDNA sequences. Maxicircles renatured as a single sequence class and had no homology with minicircles. Total minicircle complexity was greater than 300 kb. Minicircle sequence organization is complex. Different minicircles have sequences in common and comprise varying fractions of the kDNA network. Transcripts of the same maxicircle restriction fragments, representing most of the maxicircle, were detected in both bloodstream and cultured procyclic form RNA. Presumptive mitochondrial ribosomal RNA coding sequences were localized to a specific maxicircle segment. No minicircle transcription was detected. All Dk mutants examined had substantial reduction of kDNA sequences. No kDNA sequences could be detected in one mutant examined in detail. Another Dk mutant was found to contain sequences homologous to kDNA. These DNA sequences had the same electrophoretic mobility as maxicircle and minicircle restriction fragments.