The molecular organization of the ribosomal DNA repeat unit in laboratory colonies of Aedes albopictus (Skuse) and Aedes aegypti (L.) and in continuous cell lines derived from these species was examined using restriction enzyme mapping and Southern blotting techniques. In the rRNA coding regions, restriction enzyme sites were conserved in Ae. aegypti and Ae. albopictus. In particular, sites that had been mapped in rDNA cloned from the Mauritius strain of Ae. albopictus mosquitoes were confirmed in cloned DNA from cultured Ae. alboptictus cells. The intergenic spacer region of the ribosomal DNA repeat unit in Ae. albopictus was longer than that in Ae. aegypti; contained multiple recognition sites for AluI, SstI, PvuI, and XhoI; and was more heterogeneous in DNA from cultured cells than in DNA from inbred laboratory mosquitoes. The rDNA copy number was 3- to 4-fold lower in cell lines from both Ae. albopictus and Ae. aegypti than in the corresponding adult mosquitoes.