Renin catalyzes the rate-limiting step in the enzymatic cascade leading to the vasoactive peptide angiotensin II. Therefore, the activity of the renin-angiotensin system in a tissue is regulated significantly at the level of transcription of the renin gene. Besides transcription factor binding sites in the promoter region, the renin genes of human and rat contain regulatory elements also in intron I. Inclusion of intron I in reporter gene constructs with the renin promoter leads to a marked down-regulation of gene expression in nonrenin expressing 293 human embryonic kidney cells but has hardly any effect in renin-expressing L8 rat skeletal myoblasts. In combination with the cytomegalovirus immediate early gene promoter, the silencing occurs in both cell lines but is less pronounced in L8 cells. By partially deleting intron I in these constructs, we describe 5 negative (I-NRE) and 2 positive (I-PRE) regulatory elements responsible for these effects. Using gel-retardation and methylation-interference assays with 293-nuclear extracts, we detected a pseudo-palindromic protein-binding sequence between position +159 and +171 relative to the transcriptional start site. Binding of transcription factors to this sequence may be important for the tissue-specific silencing of the renin gene outside the juxtaglomerular cells of the kidney.