Previous work by our laboratory has demonstrated a pivotal role for the voltage-gated Shaker potassium channel (Kv1.3), highly expressed in the olfactory bulb (OB), in acuity, threshold, and odorant discrimination (Fadool et al. 2004). The insulin receptor (IR) kinase is expressed at high levels in the mouse OB where it is found to suppress 36 ± 4% of Kv1.3 current via tyrosine phosphorylation of critical N and C terminal residues (Fadool et al. 1998). We now show using patch-clamp electrophysiology that the adaptor protein post-synaptic density 95 (PSD-95) disrupts insulin-evoked Kv1.3 current suppression and restores peak current amplitude to 98 ± 5% of initial peak current. PSD-95 also exhibits strong modulatory effects alone on mouse Kv1.3 channel inactivation kinetics by speeding the channel inactivation by 52 ± 4%. We used immunocytochemistry and confocal microscopy to show that all three proteins are co-localized in the mouse OB, with PSD-95 showing heavy labelling across all neurolamina including the glomeruli. Using SDS-PAGE and Western blot we found that PSD-95 co-immunoprecipitates with Kv1.3, as well as the IR kinase, demonstrating a multiple protein-protein interaction in the heterologous expression system of human embryonic kidney (HEK293) cells. Confocal imaging revealed PSD-95 clusters Kv1.3 in transfected HEK293 cell membranes, as well as clusters the IR kinase, but only in the presence of Kv1.3. A PSD-95 mutant lacking the SH₃ and guanylate kinase (GK) domain (PSD-95 ΔSH3) was constructed by cDNA truncation of PSD-95, as well as use of previous mutants created by El-Husseini et al. (2000) to dissect the interaction of these three proteins through co-immunoprecipitation and confocal microscopy. The PSD-95 ΔSH3 mutant fails to cluster the IR kinase with Kv1.3 in HEK293 cell membranes, suggesting the IR kinase-PSD-95 protein-protein interaction is mediated by the PSD-95 SH₃ domain. PSD-95 ΔPM (minus palmitoylation mutant) was also used to demonstrate that PSD-95 may be involved in the trafficking and distribution of Kv1.3 by visualizing channel membrane and cytosol distribution in transfected HEK293 cells using confocal microscopy. We propose a model of interaction of Kv1.3, IR kinase, and PSD-95 where Kv1.3 channels are bound by PDZ domains 1 and 2 of PSD-95 and the IR kinase is bound by the SH₃ domain. These data suggest that PSD-95 may influence the excitability of synaptic connections in the mouse OB via K channel interaction and subsequent modulation.