Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3’ position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the carboxyl terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK C2 domain has a typical anti-parallel b-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid-binding properties of the mCPK C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to phosphatic acid (PA) and that two lysine residues, K1432 and K1434, are responsible for the PA-binding affinity.