The I domains of the leukocyte beta 2 integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp140 and Ser142, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of alpha M beta 2. Presently, we evaluated the role of two I domain regions in alpha M beta 2 ligand recognition: 1) the conserved cluster of oxygenated residues (Asp134, Asp140, Ser142, and Ser144) and 2) a 7-amino acid region (Phe246-Tyr252), conserved in alpha M and alpha X but absent in alpha L of the beta 2 integrins. Recombinant alpha M beta 2 was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp140, Asp140/Ser142, Ser142, or Ser144 produced a complete loss in the capacity of alpha M beta 2 to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp248 or Tyr252 or deletion of Phe246-Tyr252 abolished iC3b ligand recognition as well as the binding of a blocking antibody. In contrast, these mutations did not affect the binding of the cation-dependent epitope. These data implicate a second region within the I domain important for alpha M beta 2 ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of alpha M beta 2.