Nitric oxide (NO) plays a key regulatory role in cardiovascular physiology due to its vasodilatory, anti-thrombotic and anti-atherogenic properties. In endothelial cells NO is synthesised by endothelial NO synthase (eNOS), which is known to associate with a number of other proteins. Our aim was to identify novel protein associations with eNOS in human umbilical vein endothelial cells (HUVEC) and to clarify the physiological role of such associations. Analysis of eNOS immunoprecipitated from lysates of HUVEC by mass spectrometry revealed a previously unrecognised association with β-catenin. This was confirmed by western blot analyses of eNOS and β-catenin immunoprecipitates, since each protein was detected in immunoprecipitates of the other (n=3). Nuclear translocation of β-catenin following eNOS activation was assessed by western blot of nuclear lysates. eNOS stimulation with adenosine, salbutamol, histamine or thrombin increased nuclear translocation of β-catenin compared to untreated controls (n=4), as did spermine NONOate or 8-bromo-cGMP (n=5). Transcriptional activity of β-catenin, a co-activator of T cell factor (TCF) / lymphoid enhancing factor (LEF) transcription factors, was assessed using a luciferase reporter assay in HUVEC transfected with TCF/LEF reporter plasmids. Spermine NONOate, 8-bromo-cGMP or sildenafil increased TCF/LEF-mediated luciferase activity relative to controls (p<0.05, P<0.01, P<0.05 respectively, n=6). The role of β-catenin in NO-cGMP signalling was examined in mouse pulmonary endothelial cells (MPECs) using an in vitro angiogenesis assay. Stimulation of NO-cGMP signalling in wild-type (WT) MPECs using vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP or sildenafil significantly increased tube length compared to untreated controls (respective means ± SEM 13922 ± 549, 15030 ± 605, 14445 ± 549, 14589 ± 796 vs. 11608 ± 971, P<0.05); these responses were not seen with MPECs lacking the β-catenin gene (β-cat-/-) (10748 ± 473, 10424 ± 1105, 9326 ± 821, 10162 ± 1015 vs. 9908 ± 918). Both types of MPECs exhibited normal angiogenic responses to basic fibroblast growth factor which acts independently of NO-cGMP signalling (WT 9240 ± 518 vs. 7715 ± 101, P<0.05; β-cat-/- 10006 ± 250 vs. 8528 ± 83, P<0.01; n=4). Spermine NONOate, 8-bromo-cGMP or sildenafil also increased VEGF expression (which is known to be NO-dependent) relative to control in WT but not β-cat-/- MPECs, as determined by western blot of cell lysates (n=4). In summary, activation of the NO-cGMP pathway induces nuclear translocation of β-catenin and increased transcriptional activity, which may be facilitated by close approximation of β-catenin with eNOS in the resting state. Additionally, β-catenin plays a key role in NO-mediated angiogenesis and may be an important contributor to NO-mediated gene transcription and endothelial cell function.