Objective. Atrap is an interacting protein of angiotensin AT1 receptors and was shown to facilitate AT1 receptor internalization in vitro. To assess the in vivo relevance of Atrap for AT1 receptor function, we generated Atrap-deficient mice by gene targeting. Methods and Results Atrap-deficient mice were viable, fertile and showed no gross abnormalities. Atrap protein in the kidney was localized to the proximal tubule with particularly intense staining in the brush border area. We found no Atrap expression in the renal vasculature or in juxtaglomerular cells. Systolic arterial blood pressure (telemetry) was elevated in Atrap-/- compared to +/+ mice averaging 120.3±0.7 vs. 112.7±1.4 mm Hg (n=5, 1816 data points determined over 72 hrs, p=0.0014). The dose-response relationship of arterial blood pressure after acute angiotensin II infusion in anesthetized mice was similar in both genotypes. Plasma renin concentration was markedly reduced in Atrap-/- compared to wild types (49.1±5 vs. 100.3±13 ng Ang I/mL/hr, n=40 and 37, respectively; p<0.001). Conversely to the in vivo situation, baseline, stimulated (forskolin), and suppressed (Ang II) renin secretion from isolated perfused kidneys was not different between genotypes. Ambient urine osmolarity was similar in Atrap-/- and +/+. Urine pH was significantly lower in Atrap-/- compared to Atrap+/+ mice, averaging 6.51±0.11 and 7.12±0.13, respectively (n=40 and 36; p=0.0008). Acute administration of the carboanhydrase inhibitor acetazolamide caused more pronounced diuresis in Atrap-/- compared to +/+ mice (1.01±0.08 vs. 0.75±0.07 mL, n=20; p=0.02). Plasma volume was increased in Atrap-/- compared to wild types averaging 3.9±0.2 and 3.1±0.1% of body weight, respectively (n=12 and 9; p=.003). Conclusion. Atrap-deficiency leads to volume expansion and hypertension, presumably due to enhanced proximal tubular reabsorption. Thus, Atrap acts as a negative regulator of AT1 receptors in the proximal tubule of the kidney.