The H⁺-K⁺-ATPase 2 (HK2) gene of renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in mIMCD3 cells and that NF-B and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HK2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HK2 gene in mIMCD3 cells in vivo. HK2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3 trans-activated an HK2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, siRNA knockdown of Sp1 inhibited endogenous HK2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HK2 promoter without altering the binding or regulatory influence of NF-B p65 and or CREB-1 on the proximal HK2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HK2 gene expression in mIMCD3 cells in vivo and in vitro.