Crosslinking of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor complexes on a variety of cells leads to their activation through the sequential triggering of protein tyrosine kinases. Recently, DAP12 has been identified as an ITAM-bearing signaling molecule that is noncovalently associated with activating isoforms of MHC class I receptors on natural killer cells. In addition to natural killer cells, DAP12 is expressed in peripheral blood monocytes, macrophages, and dendritic cells, suggesting association with other receptors present in these cell types. In the present study, we report the molecular cloning of the myeloid DAP12-associating lectin-1 (MDL-1), a DAP12-associating membrane receptor expressed exclusively in monocytes and macrophages. MDL-1 is a type II transmembrane protein belonging to the C type lectin superfamily and contains a charged residue in the transmembrane region that enables it to pair with DAP12. Crosslinking of MDL-1/DAP12 complexes in J774 mouse macrophage cells resulted in calcium mobilization. These findings suggest that signaling via MDL-1/DAP12 complexes may constitute a significant activation pathway in myeloid cells.