Adhesion of Mycoplasma pneumoniae and the closely related M. genitalium to HEp-2 cells was investigated. The main surface proteins known to be involved in adhesion are P1 of M. pneumoniae and its homologue, MgPa, of M. genitalium. Both proteins are also immunodominant proteins. Protein P116 is another immunodominant protein of M. pneumoniae. These immunogenic proteins were investigated for their surface exposure and involvement in adhesion to host epithelial cells. Immunofluorescence microscopy (IFM) was used to detect M. pneumoniae and M. genitalium adhering to HEp-2 cells. Monospecific antibodies were produced against fragments of the surface proteins lacking tryptophan stop codons and were used for adhesion detection, surface exposure and adhesion inhibition IFM assays. Three monospecific antibodies were made against MgPa covering regions in the N-terminal, the middle and the C- terminal part; two monospecific antibodies were produced against P1 covering regions of the N- and the C-terminal part and one monospecific antibody was made against most of P116. Only the C-terminal parts of P1 and MgPa were surface exposed and blocking of these regions with the monospecific antibody resulted in inhibition of cytadsorption. Protein P116 was shown to be surface exposed and an essential protein involved in adhesion because the anti-P116 antibody prevented attachment of M. pneumoniae to the HEp-2 cells independently of P1. This study adds to the understanding of the molecular biology of M. genitalium and M. pneumoniae and presents a method to study the proteins involved in adhesion of these mycoplasmas.