Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are cotranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5' and 3' non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.