Neuronal interleukin-16 (NIL-16) is a multi-PDZ domain protein expressed in post-mitotic neurons of the hippocampus and cerebellum. NIL-16 contains four PDZ domains, two of which are located within the neuron-specific N-terminal region. In yeast two-hybrid systems, the N-terminus of NIL-16 interacts with several ion channel proteins, including the Kv4.2 subunit of A-type K(+) channels. Here we provide evidence that NIL-16, through interactions with Kv4.2, influences Kv4.2 channel function and subcellular distribution. Specifically, coexpression of NIL-16 with Kv4.2 in COS-7 cells results in a significant reduction in whole-cell A-type current densities; however, when the Kv4.2 PDZ-ligand domain is mutated, Kv4.2 current densities are not affected by NIL-16 coexpression. Moreover, single-channel conductance was not influenced by the presence of NIL-16. In hippocampal neurons, A-type current densities are increased by conditions that inhibit interactions between NIL-16 and Kv4.2, such as overexpression of the Kv4.2 C-terminal PDZ-ligand domain and treatment with small-interfering RNA duplexes that reduce NIL-16 expression. Results of surface biotinylation assays using COS-7 cells suggest that Kv4.2 surface expression levels are reduced by coexpression with NIL-16. In addition, coexpression of NIL-16 with Kv4.2 induces Kv4.2 to form dense intracellular clusters; whereas without NIL-16, Kv4.2 channels cells are dispersed. Taken together, these data suggest that interactions between Kv4.2 and NIL-16 may reduce the number of functional Kv4.2-containing channels on the cell surface. In summary, NIL-16 may provide a novel form of A-type K(+) channel modulation that is localized specifically to neurons of the hippocampus and cerebellum.