Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.