Such phagocytic leukocytes as macrophages and neutrophils are the key cellular components of innate immunity. The actin cytoskeleton is essential for their recruitment and activation in infected tissues. We have previously identified p65/L-plastin with Ca(2+)-, calmodulin-, and beta-actin-binding domains in macrophages. In order to further investigate the p65/L-plastin-involved cellular functions, we cloned the cDNA for murine grancalcin, a possible binding partner of p65/L-plastin. According to the sequence, grancalcin is a member of the penta-EF-hand protein family. We prepared recombinant (r) grancalcin for functional studies and found that it exhibited Ca(2+)-dependent precipitation. High-titer antibodies against the protein enabled us to detect intracellular grancalcin. A flow cytometric analysis revealed grancalcin to be highly expressed in macrophages and neutrophils. The protein was particularly abundant in those cells recovered from bacteria-infected sites. Immunohistochemical studies clarified that grancalcin was translocated to the actin cytoskeleton in macrophages upon exposure to bacterial lipopolysaccharide. These findings suggest that grancalcin plays a key role in leukocyte-specific functions that are responsible for host defense.