A growing body of evidence implicates ₁-adrenergic receptors (₁ARs) as potent regulators of growth pathways. The three ₁AR subtypes (_1aAR, _1bAR, _1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell-specific regulation of the human _1dAR subtype, we cloned and characterized the _1dAR promoter region in two human cellular models that display disparate levels of endogenous _1dAR expression (SK-N-MC and DU145). Results reveal that _1dAR basal expression is regulated by Sp1-dependent binding of two promoter-proximal GC boxes, the mutation of which attenuates _1dAR promoter activity 10-fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with _1dAR promoter methylation-dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation-dependent disruption of Sp1 binding in a cell-specific manner resulting in repression of basal _1dAR expression.--Michelotti, G. A., Brinkley, D. M., Morris, D. P., Smith, M. P., Louie, R. J., Schwinn, D. A. Epigenetic regulation of human _1d-adrenergic receptor gene expression: a role for DNA methylation in Sp1-dependent regulation.