Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.