PDZ domains mediate protein interactions primarily through either classical recognition of carboxyl-terminal motifs or PDZ/PDZ domain associations. Several studies have also described internal modes of PDZ recognition, most of which depend on beta-finger structures. Here, we describe a novel interaction between the PDZ domain of nNOS and Vac14, the activator of the PtdIns(3)P 5-kinase PIKfyve. Binding assays using various Vac14 deletion constructs revealed a beta-finger independent interaction that is based on a novel internal motif. Mutational analyses reveal essential residues within the motif allowing us to define a new type of PDZ domain interaction.