The number and organization of ribosomal RNA (rRNA) genes in the genome of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species were studied by the Southern hybridization technique. Restriction endonuclease-digested DNAs of the organisms were hybridized with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and with a recombinant plasmid pMC5 constructed of pBR325 and an insert containing M. capricolum genes for 23S, 5S and most of the 16S rRNA gene. The hybridization data indicate the presence of only one or two sets of rRNA genes in the mollicutes tested, a number lower than in eubacteria. The rRNA genes in mollicutes appear to be organized as clusters (acting apparently as operons) in the typical prokaryotic fashion, 5'-16S-23S-5S-3'. Despite the marked sequence homology shared by the rRNA operons of the different mollicutes and of E. coli, the operons are not identical. Thus, there is an EcoRI restriction site in the 16S rRNA genes in only 8 of the 13 species tested. The recombinant plasmid pMC5 has provided a sensitive probe for detection and identification of mollicutes in contaminated cell cultures. The purified DNA of the tested cell culture, or its supernatant fluid, was digested by EcoRI, and Southern blot hybridization of the products was performed with nick-translated pMC5. The probe did not hybridize with eukaryotic DNA. Each of the mollicutes species examinated exhibited a species-specific hybridization pattern. The hybridization tests enabled the identification of the four most prevalent mycoplasma contaminants of cell cultures, M. orale, M. hyorhinis, M. arginini and A. laidlawii. The test is capable of detecting 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. The possibility of using this approach for detection and identification of noncultivable mycoplasmas in plant and insect tissues is under investigation.