Estrogen receptor α (ERα) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ERα activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel α subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ERα also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hERα-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC(50), 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hERα-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hERα or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 μM PP2, and reported downstream ERK with 25 μM PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hERα-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 μM LY294002 decreased estrogen-stimulated hSlo1 transcription by ∼40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hERα-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.