Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.