We have compared the structure of macronuclear DNA molecules that contain rRNA genes of four hypotricous ciliates, Stylonychia pustulata, Euplotes aediculatus, Oxytricha fallax and Oxytricha nova. The macronuclear rDNA, like all macronuclear DNA in hypotrichs, exists as achromosomal molecules of approximately single-gene size. The rDNA molecules have been cloned intact as recombinant plasmids and analyzed by restriction mapping and Southern hybridization. The sites of restriction enzymes BamHI, EcoRI, HindIII, PstI, PvuII and XhoI have similar but not identical patterns in Stylonychia and the two Oxytricha rDNAs. The restriction pattern of Euplotes rDNA is unlike those of the other three, with only one site of seventeen in the same position. Despite this divergence in nucleotide sequence, the overall structure of the rDNA molecules in the four hypotrichs is constant. The size of all the rDNA molecules is the same, 7.49 kb. Also, the positions of the regions coding for 19S and 25S rRNA are alike. The 25S coding region is at the 5' end of the DNA template strand (3' end of the RNA transcript), within 500 base pairs of the terminus of the DNA molecule. The 19S coding region is adjacent to the 25S region with less than 500 base pairs of spacer lying between the two genes. The largest non-coding sequence is at the 3' end of the DNA molecule adjoining the 19S RNA gene. The 3' non-coding regions show greater sequence divergence among the different rDNAs than do the coding regions. The similarity in size and organization of these molecules and the variability in the restriction patterns suggest that the gene structure is under tighter evolutionary constraint than is the primary nucleotide sequence.