The respiratory burst of neutrophils stimulated by chemotactic factors is markedly augmented by Mac-1-dependent adhesion such as the interaction of Mac-1 (CD11b/CD18) with intercellular adhesion molecule-1 (ICAM-1; CD54) expressed on the surface of parenchymal cells (e.g., cardiac myocytes). In the current study, we evaluate the hypothesis that lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) can also trigger the respiratory burst in neutrophils. To isolate LFA-1/ICAM-1 interactions from Mac-1/ ICAM-1 interactions, full-length chimeric ICAM-1 was developed and expressed in L cells with domains 1 and 2 from canine ICAM-1 and domains 3-5 from human ICAM-1 (C1,2;H3-5). We have shown that canine neutrophils do not bind to human ICAM-1. We demonstrated that chimeric ICAM-1 C1,2;H3-5 supported only LFA-1-dependent adhesion of canine neutrophils and that such adhesion triggered rapid onset of H2O2 production from canine neutrophils. The following seven experimental conditions distinguished LFA-1-dependent H2O2 production from Mac-1-dependent production: It did not require exogenous chemotactic stimulation; H2O2 release was more rapid, but the amount released was <40% of that mediated by Mac-1 adhesion; it was inhibited by anti-CD11a and anti-ICAM-1 antibodies; in contrast to that mediated by Mac-1, it was not inhibited by anti-CD11b antibody, neutrophil inhibitory factor (NIF), or cytochalasin B or H7. Thus, canine neutrophils seem to be able to utilize two members of the beta2 integrin family to interact with ICAM-1 and signal H2O2 production, with LFA-1 at an early stage without prior chemotactic stimulation and Mac-1 at a later stage requiring chemotactic stimulation.