Uninephrectomy (UNX) causes hyperfiltration in the contralateral kidney. As does renal hypertension, where we have recently shown that increased ANGII induced nitric oxide (NO) release is associated with up-regulation of several L-arginine metabolising enzymes. The aim of the present study was to investigate NO release from afferent arterioles (AAs) and the regulation of L-arginine metabolism at the molecular level in isolated preglomerular vessels from uninephrectomised rats after two days and after six weeks. The NO sensitive fluorescent dye DAF-FM showed a baseline NO release in UNX rats, very pronounced at two days and still present after six weeks. ANGII was able to induce NO synthesis after two days of UNX, while after six weeks there was no further effect. NO synthesis was not detected in control vessels. On the molecular level, mRNA expressions of eNOS and the ANGII AT1b and AT2 receptors were decreased after two days UNX and normalised after six weeks. CAT-1, CAT-2, arginase-2, DDAH-1 and DDAH-2, caveolin-1, ANGII AT1a receptor, VEGF and COX-2 expressions were increased after two days UNX and normalised after six weeks. The protein expression of eNOS, arginase-1 and DDAH-2 was not different from controls after six weeks of UNX. In conclusion, UNX is associated with increased vascular NO release during the early phase of hyperfiltration. The increased expression of the L-arginine transporters CAT-1 and CAT-2 may increase the NO synthesis capacity and increased DDAH-1 and DDAH-2 that may reduce the endogenic inhibition of eNOS by ADMA. After six weeks the NO release is only slightly increased and vascular gene expression normalised, indicating that structural changes may be more important at this later stage.