Seventeen strains of mycoplasmata representing 11 named species were compared serologically by three parameters: growth inhibition on agar, double immunodiffusion, and complement fixation. In growth-inhibition studies, a strain labeled Mycoplasma histotropicum was found related to and perhaps best classified as M. pulmonis, a relationship confirmed by double immuno-diffusion studies. A comparison of the remaining 10 species demonstrated that two pairs of species could be shown to be closely related by complement fixation and double immunodiffusion but not by growth inhibition; these were: M. granularum-M. laidlawii and M. felis-M. canis. M. pneumoniae and M. gallisepticum were the most serologically unique organisms in this study, showing very few cross-reactions with each other or other species. Overall, taxonomic groupings obtained by comparative serology appeared to correlate with the groupings obtained when the guanine plus cytosine contents of the deoxyribonucleic acid of mycoplasmata were employed as classification criteria. The group of organisms having a guanine plus cytosine content of 23 to 28% (M. canis, M. fermentans, M. hyorhinis, M. neurolyticum, and M. pulmonis) appeared to be generally serologically related. Thus the remarkable heterogeneity observed in the base composition of the deoxyribonucleic acid of order Mycoplasmatales is also reflected and apparently paralleled by a corresponding serological heterogeneity.