Inositol polyphosphate 4 phosphatases (IP4Ps) are enzymes involved in the regulation of phosphoinositide 3-kinase lipid signaling. They catalyze the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate. In this paper we have characterized a lipid binding C2 domain located on the N-terminus of type I INN. Mutational analysis of the lipid binding loops suggests that Asp61, Asp120, Asp123, Lys122, Arg124 are involved in lipid binding in vitro. In addition, we show that this C2 domain binds calcium but calcium is not involved in lipid binding. This paper provides insight into the mechanism of membrane interaction of INN. (c) 2007 Elsevier Inc. All rights reserved.