The human CD99 protein is expressed on many cell types and is mostly abundant on lymphocytes and on several tumors. Different functions were attributed to the CD99 receptor, including adhesion, apoptosis and activation. However, until now the only ligand suggested to be recognized by CD99 was CD99 itself. In order to identify possible new CD99 ligands we constructed a CD99 protein fused to human IgG1. Surprisingly, a pronounced specific staining of melanoma cell lines that were infected with mycoplasmas was observed whereas clean cells were not recognized. Staining was specific, as other fusion proteins did not recognize the mycoplasma-infected cells. Sequencing of the 23s-16s region revealed that the contaminating agent is Mycoplasma hyorhinis. The CD99 interaction with M. hyorhinis was direct since it was blocked by anti-CD99 monoclonal antibody and by M. hyorhinis. It was also strain-specific as other mycoplasmas were not recognized. Our results show that CD99 interacts with a novel ligand of M. hyorhinis.