Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93delta. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93delta expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93delta, and association between Kir2 channels and PSD-93delta was confirmed further by the ability of anti-Kir2.1 antibodies to coimmunoprecipitate PSD-93delta from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93delta had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93delta is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system.