Engulfment of foreign pathogens is an evolutionary ancient host cell endocytic response. Signaling pathways effecting phagocytosis are divergent and largely depend on the structural features of the cell surface receptor utilized. CEACAM3, a member of the CD66 complex on human neutrophils, has been implicated as a cellular receptor promoting phagocytosis of microorganisms. The cytoplasmic domain of CEACAM3 (CEACAM(cyt)) contains an immunoreceptor tyrosine-based activation motif. In this study we demonstrate that CEACAM(cyt) is phosphorylated by protein kinase C, casein kinase I, and Src-kinase in vitro. To identify molecules binding to CEACAM(cyt) in vivo, we used differentially phosphorylated recombinant expressed CEACAM cytoplasmic domains to isolate CEACAM(cyt)-associated proteins from granulocyte extracts. Calprotectin, which modulates neutrophil integrin-mediated adhesion and leukocyte trafficking and displays antimicrobial activity, interacts specifically with CEACAM(cyt). This interaction is calcium-modulated but independent of phosphorylation of CEACAM(cyt) Although tyrosine-phosphorylated CEACAM(cyt) binds and stimulates Src-kinases in vitro, no CEACAM(cyt)-associated phosphokinase activity was copurified.