Transcription of the mitochondrial genome in Dictyostelium discoideum gives rise to eight major polycistronic RNA species that can be detected by Northern hybridization. In order to determine whether these transcripts could possibly derive from processing of even larger transcripts, reverse transcriptase polymerase chain reactions (RT-PCRs) were performed in an attempt to amplify the intervening regions between the eight major transcripts. All but one intervening region were successfully reverse transcribed and amplified, indicating that even larger transcripts existed and that the eight major transcripts detected previously may be the products of transcript processing. Southern hybridization analyses of DNA fragments representing the sequences between the eight major transcripts with in vitro capped mitochondrial RNA identified the 5′ end of only one of the eight major transcripts as a genuine transcription start site. The ability to initiate transcription from DNA sequences upstream of the identified transcription initiation site was demonstrated in bacterial cells expressing the Dictyostelium mitochondrial RNA polymerase. We conclude that transcription of the Dictyostelium mitochondrial genome is initiated at a single site, generating a large polycistronic transcript that is very efficiently, probably cotranscriptionally processed into mature RNA species. This is the first report on a protist mitochondrial DNA that is, although much larger in size than its metazoan counterparts, transcribed from a single transcription initiation site.