It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniae is a main cross-reactive antigen with similar molecular weight protein of Mycoplasma genitalium by Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniae and hyperimmune rabbit sera directed against each mycoplasma strain.Mycoplasma genitalium and Mycoplasma pneumoniae are genetically (4, 19) and serologically (2, 3, 9-11, 16, 20) close-related organisms that colonize in humans. M. genitalium is rarely isolated from human urethral specimens (12, 18) and recently from throat specimens of military recruits (1), but the pathogenicity of this organism has not been assessed. Mycoplasma pneumoniae is a human pathogen causing upper respiratory infections and atypical pneumonia, especially in children. Both species have several common features such as glucose fermentation, complex nutritional requirements, and adherence of erythrocytes to the colonies. Genomic homology of DNAs between both strains analyzed by DNA-DNA hybridization was only 1.8 to 8% (10, 19), suggested that M. genitalium might have evolved independently of other human mycoplasma species, including M. pneumoniae (5). It has been demonstrated that monoclonal antibody (MAb) specific to the P1 protein (168 kDa) which is the major adhesin of M. pneumoniae cross-reacted with 100 kDa protein which is a putative adhesin of M. genitalium (3). Furthermore, Hirschberg et al (7) showed that MAbs directed against M. pneumoniae proteins of 200, 170, 67, 47, and 42 kDa cross-reacted with M. genitalium proteins of 94, 145, 63, 44, and 38 kDa, respectively. In the present study, we showed that a 67 kDa protein of M. pneumoniae was a predominant antigen that cross-reacted with a similar molecular weight protein of M. genitalium using hyperimmune rabbit sera to M. genitalium and to M.pneumoniae and MAb specific to 67 kDa protein of M. pneumoniae by Western blotting.M. pneumoniae FH was from our laboratory stock and M. genitalium G37 was