Adhesins on the surface of mycobacteria may contribute to the establishment of infection by promoting bacterial attachment to host tissues. A number of bacterial adhesins with lectin characteristics display hemagglutination (HA) activity and can be purified based on their affinity for heparin. In an attempt to identify mycobacterial adhesins, cell lysates of heattreated mycobacteria were phaseseparated by Triton-X114 and chromatographed on a heparin Sepharose column. HA activity was found primarily in the aqueous (hydrophilic) phase and was eluted from the column with 0.3 to 0.5 M NaCl. Monoclonal antibody D2, which reacted (via immunoblots) with a major protein band of 27 kDa that is present in the heparin eluted fractions, specifically inhibited HA activity. This antigen is a mycobacterial cell surface component as shown by immunogold electron microscopy. The HA activity requires the presence of a protein component on the surface of red blood cells since it is eliminated by pronase treatment of the cells whereas sialidase treatment has no effect. Interestingly, the heparin binding hemagglutinin also induces bacterial aggregation when added to a population of dispersed mycobacteria. These results suggest that the heparin binding hemagglutinin is a cell surface protein of mycobacteria with adhesin characteristics that may play a role in mycobacterial colonization of host tissues.