Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.