Early in their developmental program, Dictyostelium discoideum exhibit EDTA-sensitive and EDTA-resistant adhesion. The molecules which mediate the adhesions have been called contact sites, with contact sites A mediating EDTA-resistant adhesion and contact sites B mediating EDTA-sensitive adhesion. The studies described here have revealed that prior to aggregation, a second EDTA-sensitive adhesion system emerges. In keeping with previously established nomenclature, the molecules mediating the newly discovered adhesion system have been called contact sites C. Unlike contact sites B, contact sites C are unaffected by a contact sites B-blocking peptide. Contact sites C-mediated adhesion is also distinct from contact sites B-mediated adhesion in that contact sites C-mediated adhesion is EGTA-resistant and in the presence of EDTA it can be rescued by the addition of Mg2+. Thus Mg2+ may be the cation present under physiological conditions that is essential for contact sites C activity. Unlike contact sites B-mediated adhesion, contact sites C-mediated adhesion is not observed in growing amoebae. Contact sites C-mediated adhesion first becomes apparent within hours after the initiation of development and its strength appears to increase throughout the first 10 h of the developmental program. A mutant lacking the EDTA-resistant contact sites A exhibits normal contact sites B- and C-mediated adhesion, demonstrating that both EDTA-sensitive adhesion systems are independent of contact sites A. Thus aggregating D. discoideum amoebae possess three distinct adhesion systems, one of them is EDTA-resistant and the other two are EDTA-sensitive.