Radiolabeled DNA probes prepared in vitro by the nick translation method were used to determine the nucleotide sequence homology among the eight established and one unclassified species of Acholeplasma. Very little DNA homology (2 to 21 percent) was found among these nine distinct species and the heteroduplexes showed at least 15 percent mismatching as determined by thermal elution endpoints. The data obtained by hybridization analyses paralleled the results obtained by the growth inhibition and epi-immunofluorescence serologic procedures. The small amount of nucleotide sequence homology among the nine distinct species indicate that the Acholeplasma species are quite distinct and unrelated to each other genomically, findings which should provide useful insight on the molecular biology and evolutionary pathways of these organisms. Labeled 3H-DNA probes to five strains of either A. laidlawii or A. axanthum hybridized to a varying degree to excess amounts of unlabeled DNAs from 12 strains of A. laidlawii and six strains of A. axanthum, respectively. Nucleic acid hybridization analyses showed a wide variation (48 to 100 percent) in DNA homologies among different strains of the two species. The results demonstrate that strains of A. laidlawii and/or A. axanthum isolated from diverse hosts and habitats (birds, rodents, cats, swine, sheep, cattle, horses, goats, primates, and plants) exhibit extensive genotypic variations. 3H-DNA-DNA hybridization procedures were found to be extremely useful in establishing or confirming the existence of distinct species within the genus Acholeplasma.