Calcium-dependent protein kinases (CDPK) are a major group of calcium-stimulated kinases found in plants and some protists. Many CDPKs are membrane-associated, presumably because of lipid modifications at their amino termini. We investigated the subcellular location and myristoylation of AtCPK5, a member of the Arabidopsis CDPK family. Most AtCPK5 was associated with the plasma membrane as demonstrated by two-phase fractionation of plant microsomes and by in vivo detection of AtCPK5-GFP fusion proteins. AtCPK5 was a substrate for plant N-myristoyltransferase and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In transgenic plants, a G2A mutation completely abolished AtCPK5 membrane association, indicating that myristoylation was essential for membrane binding. The first sixteen amino acids of AtCPK5 were sufficient to direct plasma membrane localization. In addition, differentially phosphorylated forms of AtCPK5 were detected both in planta and after expression of AtCPK5 in a cell-free plant extract. Our results demonstrate that AtCPK5 is myristoylated at its amino terminus and that myristoylation is required for membrane binding.