In cardiac cells, KCNQ1 assembles with KCNE1 and forms a channel complex constituting the slow delayed rectifier current I(Ks). Expression of KCNQ1 and KCNE1 are regionally heterogeneous and changes with pathological states of the heart. The aims of this study were to decipher the molecular mechanisms for transcriptional and post-transcriptional regulation expression of KCNQ1 and KCNE1 genes and to shed light on the molecular mechanisms for their spatial heterogeneity of distribution. We cloned the 5'-flanking region and identified the transcription start sites of the KCNQ1 gene. We characterized the core promoters of KCNQ1 and KCNE1 and revealed the stimulating protein (Sp1) as a common transactivator of KCNQ1 and KCNE1 by interacting with the Sp1 cis-acting elements in the core promoter regions of these genes. We also characterized the 3' untranslated regions (3'UTRs) of the genes and experimentally established KCNQ1 and KCNE1 as targets for repression by the muscle-specific microRNAs miR-133 and miR-1, respectively. We demonstrated spatial heterogeneity of KCNQ1 and KCNE1 distributions at three axes (interventricular, transmural and apical-basal) and disparity between mRNA and protein expressions of these genes. We also found characteristic regional differences of expressions of Sp1 and miR-1/miR-133 in the heart. Our study unraveled a novel aspect of the cellular function of miRNAs and suggests that the I(Ks)-encoding genes KCNQ1 and KCNE1 expressions are dynamically balanced by transcription factor regulation and miRNA repression. The heterogeneities of Sp1 and miR-1/miR-133 offer an explanation for the well-recognized regional differences and disparity between mRNA and protein expressions of KCNQ1 and KCNE1.