Calcium-independent phospholipase A2 (iPLA2) is the major phospholipase A2 activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA2β (riPLA2β underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA2β (83 kDa) in the presence of EGTA alone, Ca+2 alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca +2 together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA2β indicated the specific protection of riPLA2β by calcium-activated CaM at a cleavage site ≈15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA2β. Second, functional characterization of products from these partial tryptic digests demonstrated that ≈90% of the 68-kDa riPLA2β tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca2+, although > 95% of the noncleaved riPLA2β as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA2β was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ( 694AWSEM-VGIQYFR705) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694-705) bound to dansylcalmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA2β by CaM.