Excess transforming growth factor-1 (TGF-1) in the kidney leads to increased cell proliferation and deposition of extracellular matrix, resulting in progressive kidney fibrosis. TGF-1, however, stabilizes and attenuates tissue injury through the activation of cytoprotective proteins, including heme oxygenase-1 (HO-1). HO-1 catabolizes pro-oxidant heme into substances with anti-oxidant, anti-apoptotic, anti-fibrogenic, vasodilatory and immune modulatory properties. Little is known regarding the molecular regulation of human HO-1 induction by TGF-1 except that it is dependent on de novo RNA synthesis and requires a group of structurally related proteins called Smads. It is not known if other DNA binding proteins are required to initiate transcription of HO-1 and, furthermore, the promoter region(s) involved in TGF-1-mediated induction of HO-1 has not been identified. The purpose of this study was to further delineate the molecular regulation of HO-1 by TGF-1 in human renal proximal tubular cells. Actinomycin D and nuclear run-on studies demonstrate that TGF-1 augments HO-1 expression by increased gene transcription and does not involve increased mRNA stability. Using transient transfection, small interfering RNA, decoy oligonucleotides and mithramycin A experiments a ~280bp TGF-1-responsive region is identified between 9.1 and 9.4kb of the human HO-1 promoter which contains a putative Smad binding element and specificity protein 1 (Sp1) binding sites, both of which are required for human HO-1 induction by TGF-1.