The overlapping ND4L and ND5 genes of Neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. All three intervening sequences are class I introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. They contain long open reading frames (ORFs; from 306 to 425 codons long) that are continuous and in frame with the upstream exon sequences. These ORFs contain the conserved decapeptide-encoding sequences that are characteristic of the ORFs present in most class I introns. Extensive homology exists among the ORFs encoded by the ND4L intron, ND5 intron 1, and the second intron of the N. crassa oli2 gene. Also, internal repeats of about 130 amino acid residues are present twice in each of these three ORFs, suggesting that a duplication event may have occurred in the formation of these ORFs. The ND4L intron shares extensive homology (at the levels of both primary and proposed secondary structures) with the self-splicing intervening sequence present in the Tetrahymena nuclear rRNA gene. This homology includes but is not limited to the core secondary structure, as peripheral structural elements are also conserved in the two introns.