To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.