Mycoplasma pneumoniae strains PI-1428, M129, B176, FH, and MAC were analyzed for homology by DNA-DNA hybridization, thermal elution midpoints of the DNA-DNA heteroduplexes, DNA cleavage patterns with restriction endonucleases, and protein gel electrophoretic profiles. These properties were compared with biological assays, such as antigenic reactivity with reference antisera in the growth inhibition and metabolic inhibition tests, as well as the ability to attach to human WiDr cell cultures. The avirulent, nonhemadsorbing strain B176 could be differentiated from the others by diminished attachment capacity to WiDr cells and by slightly reduced DNA homology. The other strains appeared to be identical when examined by these procedures. No significant differences were detected among any of the strains by the metabolic inhibition or growth inhibition tests. The DNA cleavage patterns of the five strains were also similar, although strains FH and MAC differed from the other three strains in a few bands. These results demonstrate that these five strains are similar and that the species M. pneumoniae is remarkably homogeneous.