Lymphocyte function-associated antigen-1 (LFA-1) is a beta 2 integrin that participates in a broad range of leukocyte functions through binding to its ligand intercellular adhesion molecule-1 (ICAM-1). The location of the ICAM-1 binding site on LFA-1 is not known. A approximately 200-amino acid "inserted" or "I" domain, which is part of the beta 2 integrin alpha subunit, is homologous to the "A" domains found in the adhesive protein von Willebrand factor and in a number of other proteins. In von Willebrand factor, the A domains are involved in ligand binding, but their function in the other proteins is still unclear. In this report, we show that the LFA-1 I domain contains a binding site for ICAM-1, which can be expressed as an isolated functional unit. The I domain contains the epitopes for 18 out of 20 anti-LFA-1 monoclonal antibodies, many of which interfere with the interaction between LFA-1 and ICAM-1. The I domain binds directly to purified recombinant ICAM-1 and also inhibits LFA-1-dependent T cell adhesion to ICAM-1. This report establishes the I domain as an ICAM-1 binding region in LFA-1 and the first ligand binding site to be identified in a beta 2 integrin.