Mechanisms for selective targeting to unique subcellular sites play an important role in determining the substrate specificities of protein kinases, Here we show that stress-activated protein kinase-3 (SAPK3, also called ERK6 and p38 gamma), a member of the mitogen-activated protein kinase family that is abundantly expressed in skeletal muscle, binds through its carboxyl-terminal sequence -KETXL to the PDZ domain of alpha 1-syntrophin, SAPK3 phosphorylates alpha 1-syntrophin at serine residues 193 and 201 in vitro and phosphorylation is dependent on binding 60 the PDZ domain of alpha 1-syntrophin, In skeletal muscle SAPK3 and alpha 1-syntrophin co-localize at the neuromuscular junction, and both proteins can be co-immunoprecipitated from transfected COS cell lysates, Phosphorylation of a PDZ domain-containing protein by an associated protein kinase is a novel mechanism for determining both the localization and the substrate specificity of a protein kinase.