S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, AECC members Bresnick and Almo established S100A4-deficient mice by gene targeting. The S100A4(-/-) mice were fertile, grew normally and exhibited no overt abnormalities; however, the loss of S100A4 resulted in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages derived from S100A4(-/-) mice displayed defects in chemotactic motility in vitro. S100A4(-/-) bone marrow macrophages formed unstable protrusions, overassembled myosin-IIA, and exhibited altered CSF-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility, demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo, and suggest that this targeting this protein could suppress tumor invasion and metastasis.