The 26,300-nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the demosponge Suberites domuncula (Olivi, 1792), the largest in size yet found in Porifera, has been determined. We describe the second hadromerid sponge mitochondrial genome that contains the same set of 41 genes as the hadromerid sponge Tethya actinia, including trnMe(cau), trnI2(cau), trnR2(ucu), and atp9, all of which are transcribed in the same direction. Furthermore, rRNA genes for the small and large ribosomal subunit are very long, rns is indeed the longest among Metazoa (1833 bp). Intergenic regions (IGR) comprise about 25% of S. domuncula mtDNA and include numerous direct and inverted repeats, as well as palindromic sequences. No overlapping genes and introns were found. Phylogenetic analyses based on concatenated amino acid sequences from twelve mitochondrial protein genes strongly support the affiliation of S. domuncula to the order Hadromerida. Moreover, we have analyzed and compared two segments of mtDNA which include the three IGR from S. domuncula (12 and 16 specimens for segments I and II) and Suberites ficus (10 and 5 for segments I and II, respectively). S. ficus has frequently been reported as being both synonymous with, as well as a separate species from S. domuncula. We have found polymorphisms in IGR of both species and long deletions (43 and 167 bp in size) in two IGR of S. ficus.