A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.