We have previously shown an enhanced expression of Gia proteins and their mRNA in hearts and aortas of spontaneous hypertensive rats (SHR) as compared to Wistar-Kyoto rats (WKY). The enhanced expression of Gia proteins precedes the development of hypertension. Oxidative stress is shown to be increased in hypertension. The present studies were undertaken to examine if oxidative stress is a contributing factor in the enhanced expression of Gia proteins in hypertensive rats. Vascular smooth muscle cells (VSMC) from 12-week old SHR and their age-mathed WKY control rats were used in the present studies. The levels of G proteins were determined by western blotting using specific antibodies against G proteins. The expression of Gia-2 and Gia-3 was significatively increased by about 25% and 30% respectively in VSMC from SHR as compared to VSMC from WKY, whereas the levels of Gsa remained unchanged. The treatment of VSCM from SHR with antioxidants such as N-acetylcysteine (NAC) and diphenyleneiodonium (DPI) restored the enhanced expression of Gia proteins towards control levels. For example, NAC, at 20 mM decreased the enhanced expression of Gia-2 and Gia-3 by about 45% and 55%, respectively. Similarly, DPI (10 µM) was able to decrease the enhanced levels of Gia-2 and Gia-3 by about 50% and 60%, respectively. In addition, VSMC from SHR showed an enhanced inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by GTPgS as compared to VSMC from WKY which was restored to WKY levels by NAC or DPI. Furthermore, angiotensin-II (AngII) and C-ANP_4-23 (a ring-deleted analog of atrial natriuretic peptide) inhibited adenylyl cyclase activity to a greater extent in VSMC from SHR as compared to VSMC from WKY and NAC or DPI were able to restore the enhanced inhibition of adenylyl cyclase to WKY levels. These results suggest that oxidative stress may be a contributing to the enhanced expression of Gi proteins and associated signalling in hypertension.