Identification of the attachment factor on virulent Mycoplasma pneumoniae organisms which permits surface parasitism of respiratory epithelium was attempted. Brief pretreatment of M. pneumoniae monolayers with protease prevented mycoplasma attachment ot sensitive host cells without reducing viability of the microorganisms. Gel electrophoretic analysis of mycoplasma proteins before and after exposure of intact mycoplasmas to protease revealed the absence of a major protein species (P1) in enzyme-treated preparations while other protein bands with the exception of P2 were virtually unaffected. The absence of P1 correlated with the failure of enzyme-treated mycoplasmas to attach to tracheal explants. P1 regeneration after protease treatment of mycoplasma monolayers was directly associated with reattachment capabilities in M. pneumoniae. Erythromycin inhibited P1 resynthesis, thus preventing resumed attachment activity by mycoplasmas. Lactoperoxidase-catalyzed iodination of intact M. pneumoniae organisms further confirmed that P1 was an external membrane protein and suggested that his surface component was required for the successful membrane-membrane interaction between host and parasite.