Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin super-family. The myristoyl group and first eight residues of Frq1 are solvent-exposed and Ca2+ binds the second, third and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (V127, A128, V131, L132, L135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (L101, W103, V125, L138, I152, L155); side chains in the Pik1 C-terminal helix (A157, A159, L160, V161, M165, M167) contact solvent-exposed residues in the Frq1 N-terminal pocket (W30, F34, F48, I51, Y52, F55, F85, L89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (8-760) from its catalytic domain (792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an auto-inhibitory constraint. Deletion of the lipid kinase unique (LKU) motif (35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the LKU motif with the kinase domain.