Human RNA helicase II/Gu (hRH II/Gu) protein unwinds double-stranded RNA, folds single-stranded RNA and may play important roles in ribosomal RNA biogenesis, RNA editing, RNA transport, and general transcription. The genomic organization and the regulation of expression of the hRH II/Gu gene were analyzed. The hRH II/Gu gene is over 29 kb in length, and includes 15 exons. All intron-egon junctions contain consensus GT-AG sequences. An 85 bp basic promoter was identified which lacks a functional TATA box. In LNCaP cells, the hRH II/Gu promoter was up to 50 times as active as pGL2-control vector which has an SV 40 early promoter. Mutation of a putative c-Myc/USF binding site in the region between -110 and -85 caused a decrease in the promoter activity by 2- to 3-fold. A negative regulatory region was also found between -324 and -264. Studies of transcriptional regulation of hRH II/Gu under different physiological and pathophysiological conditions may provide clues to its biological function.