Similarities in the biochemical and serological characteristics of sterol-nonrequiring acholeplasmas (mycoplasmas) have created problems in the taxonomy and classification. The present study was designed to compare the genetic characteristics of two unclassified acholeplasma strains with those of the established speciesAcholeplasma. Radiolabeled DNA probes were prepared toA. laidlawii, A. granularum, and to the two unclassified acholeplasmas (S2 and 72-043 strains) by the nick-translation method. Nucleic acid hybridization results presented here confirm and extend previously reported genetic interrelationships observed betweenA. laidlawii (PG8) andA. granularum (BTS39). The DNA of theseAcholeplasma species failed to hybridize with DNA prepared from aMycoplasma species (M. capricolum, California kid strain) or from duck cells in control studies. The two unclassified acholeplasmas were found to be related to each other but distinct from all of the established species ofAcholeplasma and may represent a new species within the genusAcholeplasma. In light of difficulties in making species separation in the acholeplasmas, data from nucleic acid hybridization tests can offer very important information useful to the classification of these organisms.