In recently published studies, we show that angiotensin II (AII) generated from an engineered rat angiotensinogen cDNA, and maintained intracellularly, is growth stimulatory for a rat hepatoma cell line. In the present study, we report that co-expression of AII fused to cyan fluorescent protein (ECFP/AII) and angiotensin type I receptor fused to yellow fluorescent protein (AT1R/EYFP) enhances proliferation of COS-7 and CHO-K1 cells by 59% and 64%, respectively, compared to cells expressing the corresponding independent proteins (P < 0.001 for both). This effect is inhibited by losartan, suggesting (as in our previous published studies) that losartan is internalized by the cells, via receptor-mediated endocytosis, and thus inhibits intracellular receptor-ligand interaction. The growth effect is independent of anti-AII antibodies suggesting that it does not reflect AII secretion into the culture media; AII is also undetectable in the media. Expression of AT1R/EYFP with ECFP/AIIC (control scrambled sequence AII fused to ECFP) has no effect upon cell proliferation. ECFP/AII also alters the cellular localization of AT1R/EYFP. ECFP/AII is concentrated in the nucleus, but shows diffuse cytoplasmic fluorescence as well. AT1R/EYFP, expressed independently, is visible in the endoplasmic reticulum and Golgi apparatus of COS-7 and CHO-K1 cells as early as 24-h post-transfection. At 72 h, it is visibly associated with the plasma membrane. By 144 h, 85% of the cells show detectable circumferential fluorescence. In contrast, in cells that express AT1R/EYFP and ECFP/AII, both proteins accumulate in the nucleus and only 13% of the cells show visible plasma membrane-associated yellow fluorescence at 144 h (P < 0.001). Furthermore, co-expression of ECFP/AII with AT1R/EYFP stimulates cAMP response element-binding protein (CREB) activity in CHO-K1 and COS-7 cells. Exogenous AII similarly significantly increases CREB activation in AT1R/EYFP-stably transfected CHO-K1 and COS-7 cells.