The H⁺-K⁺-ATPase α₂ (HKα₂) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the κB DNA binding element at −104 to −94 influences basal HKα₂ gene transcription in these cells. Recombinant NF-κB p50 footprinted the region −116/−94 in vitro. Gel shift and supershift analysis revealed NF-κB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated −104/−94 NF-κB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-κB p50, p65, or their combination trans-repressed the HKα₂ promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-κB-mediatedtrans-repression of the HKα₂ promoter. HDAC6 overexpression inhibited HKα₂ promoter activity, and HDAC6 coimmunoprecipitated with NF-κB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-κB to suppress HKα₂ transcription and identify NF-κB p50 and p65 as novel binding partners for HDAC6.