Angiotensin (Ang) AT1 receptors and Ang-converting enzymes (ACE and ACE2) are expressed in the dorsal vagal complex (DVC) of the brainstem. The aim of this study was to examine in vivo interactions between brainstem Ang AT1 receptors, ACE and ACE2 using small, hairpin RNA (shRNA) gene-silencing methods. The study takes advantage of the bilateral brainstem expression of renin-angiotensin system (RAS) markers. Adenovirus vectors (Ad, 2.0 x 10(9) c.f.u. ml(-1), 200 nl) carrying interference small hairpin RNA (shRNA) for either AngAT1a (Ad-AT1a-shRNA) or AngAT1b (Ad-AT1b-shRNA) were microinjected into the right side of the brainstem DVC. The Ad-LacZ control was injected into the left side. Brainstems were processed with in situ hybridization and immunochemistry. Results showed that: (1) Ad-AT1a-shRNA downregulated Ang AT1a mRNA by 61.2 +/- 6.8% (P < 0.01) and Ad-AT1b-shRNA downregulated Ang AT1b mRNA by 51.6 +/- 5.2% (P < 0.01); (2) downregulation of Ang AT1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 +/- 14.5%, P < 0.01), while reduction in Ang Ad-AT1b mRNA had no effect; (3) ACE mRNA expression was not altered by either RNA interference (RNAi) treatment; and (4) immunochemical staining for Ang AT1 receptors, ACE and ACE2 were in agreement with the mRNA changes observed. These results demonstrate the utility of in vivo gene silencing to examine functional specificity. Both Ad-AT1a-shRNA and Ad-AT1b-shRNA induced site- and subtype-specific downregulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT1a receptors and the RAS regulatory enzyme, ACE2.