Tartrate-resistant acid phosphatase (TRAP) is a lysosomal di-iron protein of mononuclear phagocytes and osteoclasts. Hitherto, no role for the enzyme in immunity has been identified; however, knockout mice lacking TRAP have a skeletal phenotype caused by an intrinsic osteoclast defect. To investigate a putative function for TRAP in macrophages (Mphi), we investigated proinflammatory responses and systemic microbial clearance in knockout mice compared with age- and gender-matched congenic wild-type mice. Phorbol 12-myristate 13-acetate (PMA)-stimulated and interferon-gamma (IFN-gamma)-induced superoxide formation was enhanced in peritoneal Mphi lacking TRAP; nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-gamma was also increased. In addition, secretion of the proinflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-12, was significantly greater in TRAP-deficient Mphi when stimulated with LPS, with or without addition of either TNF-alpha or IFN-gamma. The activity of tartrate-sensitive (lysosomal) acid phosphatase was increased in Mphi from the knockout mice but activities of the lysosomal hydrolases N-acetyl beta-glucosaminidase and acid beta-glucuronidase were unchanged, indicating selective activation of compensatory acid phosphatase activity. Evidence of impaired Mphi function in vivo was obtained in TRAP knockout mice, which showed delayed clearance of the microbial pathogen, Staphylococcus aureus, after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates obtained from TRAP knockout mice had a reduced population of Mphi. As peritoneal Mphi and neutrophils lacking TRAP were able to phagocytose and kill S. aureus normally in vitro, TRAP may directly or indirectly influence recruitment of Mphi to sites of microbial invasion. Our study shows that TRAP participates in the inflammatory response of the Mphi and influences effector signalling pathways in innate immunity.