Mechanical forces contribute for maintenance of cardiovascular homeostasis via the control of release and production of vasoactive substances. We demonstrated previously that shear stress decreases rat ACE activity and expression. Using a reporter gene approach and mutagenesis we show now that the classical SSRE (GAGACC) contained within 1274 bp of this promoter is not functional in response to shear stress (15 dynas/cm², 18 hs) (WLuc - C:107±6.5%, SS: 65.9±9.4%, N=8; WSS-mut - C: 100±8.2%, SS: 60.2±5.2%, N=10, respectively). Analysis of progressive deletion mutants unraveled a 57bp fragment, position -251 to -194 from the transcription start site, containing functional SSRE (WLuc - C:107±6.5%, SS: 65.9±9.4%, N=8; 378 - C:100±6.4%, SS: 60.4±4.3%, N=11; 251 C: 99.7±2.6%, SS: 63.2±5.5%, N=7; 194 - C: 104.6±8.1%, SS: 92.4±6.9%, N=9). This fragment responded to shear stress even in the context of a heterologous promoter. Finally, functional analysis of mutated candidate regulatory elements identified by gel shift, DNase I footprint, and conservation of aligned sequences, revealed that only the double mutant (Barbie/GAGA-mut) but not isolated disruption of the Barbie- (WBarbie-mut) or the GAGA- (WGAGA-mut) prevented the shear stress induced response (Barbie/GAGA-mut: C: 97.9±5%, SS: 99.4±7.2%, N=6; WBarbie-mut - C: 106.1±8.6%, SS: 65.9±9.4%, N=6; WGAGA-mut - C: 100.1±2.9%, SS: 66.7±1.6, N=6;). Taken together, these data provide direct evidence for the new role of Barbie- and GAGA-boxes in mediating the shear stress induced downregulation of rat ACE expression and demonstrate that the classical SSRE (GAGACC) is not functional under the experimental conditions tested.