Mycoplasmas associated with or isolated from tissues of AIDS and non-AIDS patients have been characterized serologically and compared to reference mycoplasmas of human origin maintained in this laboratory. In collaborative studies using both serologic and genetic markers, we were able to provide specific identification of the organisms as to Mycoplasma species. Several strains of M.fermentans were identified. The new M.fermentans strains isolated from humans or from cell cultures were genetically and serologically homogenous to the type strain (PGI8) isolated over 30 years ago from the human urogenital tract. At least two isolates of M. pirum were identified through serologic and biochemical markers. Both strains had been cultivated initially by a collaborating laboratory from primary leukocyte cultures of AIDS patients, employing SP-4 mycoplasma culture medium developed earlier in this laboratory. This was a significant observation, since all previous isolations of this species had been from cell cultures and there existed no well documented information on the possible host origin of this mycoplasma. M. genitalium was also identified by the collaborating laboratory in a washed erythrocyte suspension from a patient with AIDS-related complex. Although attempts to culture the organism from the primary erythrocyte material was unsuccessful, DNA implication tests using primers specific for the organism (and capable of differentiating M. pneumoniae) were positive for M. genitalium. Current attempts are underway to cultivate the organism from this and other AIDS-derived clinical material.