We recently reported that human renin gene transcription is stimulated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma in the renin-producing cell line Calu-6. The effect of PPARgamma was mapped to two sequences in the renin promoter a direct repeat hormone response element (HRE), which is related to the classical PPAR response element (PPRE) and a nonconsensus palindromic element with a 3-bp spacer (Pal3). We now find that PPARgamma binds to the renin HRE. Neither the human renin HRE nor the consensus PPRE was sufficient to attain the maximal stimulation of renin promoter activity by the PPARgamma agonist rosiglitazone. In contrast, the human renin Pal3 element mediates both the full PPARgamma-dependent activation of transcription and the PPARgamma-driven basal renin gene transcription. The human renin Pal3 sequence was found to selectively bind PPARgamma and the retinoid X receptor-alpha from Calu-6 nuclear extracts. This is in contrast to the consensus PPRE, which can bind other nuclear proteins. PPARgamma knockdown paradoxically did not attenuate the stimulation of the endogenous renin gene expression by rosiglitazone. Similarly, a deficiency of PPARgamma did not attenuate the activation of the minimal human renin promoter, which contains the endogenous Pal3 motif. However, when the human renin Pal3 site was replaced by the consensus PPRE sequence, PPARgamma knockdown abrogated the effect of rosiglitazone on renin promoter activity. Thus, the human renin Pal3 site appears to be critical for the PPARgamma-dependent regulation of gene expression by mediating maximal transcription activation, particularly at the low cellular level of PPARgamma.