The spacer sequences of rDNAs of members of the melanogaster species subgroup of Drosophila (melanogaster, simulans, mauritiana, teissieri, and yakuba) have been compared. The external transcribed spacers (ETSs; the region encoding the 5' end of the primary transcript, upstream from the 18S sequences) are highly conserved between in D. melanogaster, D. simulans and D. mauritiana, whereas the more distantly related D. yakuba and D. teissieri differ in having apparent deletions of 22 and 27 bp, respectively, in this region. The divergence of nucleotide sequence upstream from the transcription start points is consistent with the established phylogeny of the five species. The sequences between bp positions -47 and +24 from the primary transcription start point show extremely little variation between each species. This is also the case for sequences between the approximate bp positions -140 to -125 and -85 to -70. This could indicate a functional importance not only of the sequences next to the transcription start point, but also of these upstream regions. An array of 240-bp repeats can be found at a comparable distance upstream from the transcription start point in each species. Matrix homology comparisons indicate that for each species not only is the sequence at the primary transcription start point duplicated within the 240-bp repeats as previously reported for D. melanogaster, but that this is part of a longer interrupted duplication which includes a region of strong similarity with the sequence between the approximate positions -105 to -65. This region is contained within one of the regions upstream from the transcription start point that is strongly conserved between the species. This sequence may therefore have functional significance not only for the transcription of the rRNA precursor, but also for transcription of the so-called NTS sequences which is now known to occur. The 240-bp arrays are themselves highly conserved within a species indicating that homogenisation mechanisms are operative. The divergence of these arrays between species is consistent with the phylogenetic tree. The 3' sequences of the primary transcription unit, now known to be RNA-processing sites, are also highly similar between the species. Immediately downstream from these sites there is little homology between the rDNA of the different species, until 95-bp tandem arrays are reached in each case.