Nitric oxide (NO) is an important anti-atherogenic factor and its production by endothelial NO synthase (eNOS) is disrupted early in plaque development. Constitutive ("basal") NO production is believed to be of highest relevance in its protective effects. We noticed, however, that basal eNOS activity does not always correlate with agonist stimulated eNOS sensitivity, pointing to different regulation mechanisms. We investigated the effects of time and kinase inhibitors. C57Bl/6 mouse (4 months) thoracic aorta segments were suspended in organ baths. Basal eNOS activity was measured by its capability to suppress phenylephrine (1mM) contractions measured at 1h intervals, followed by dose-relaxation curves to acetylcholine. Responses in the presence of NOS inhibitors served as control. Afterwards, phosphorylation of eNOS at S1177 and T495, and Akt at S473 was investigated using Western blot. Phenylephrine contractions increased gradually in time (1.1±0.6, 4.4±1.1 and 7.9±0.8 mN, resp. at 1, 2 and 3h; n=4), suggesting decreased basal NO production, while acetylcholine sensitivity remained unaltered. Western blot showed a loss of Akt phosphorylation (-49±16%) after 2h without changes in eNOS phosphorylation. The phosphoinositide 3 kinase (PI3K) inhibitor wortmannin raised phenylephrine contractions (+57±7%; n=4 at 1h), without changing sensitivity to acetylcholine or exogenous NO (DEANO), or smooth muscle function. This effect was abolished by preincubation with the tyrosine kinase inhibitor erbstatin. Finaly, wortmannin strongly inhibited Akt phosphorylation (­97±3%), but did not alter eNOS phosphorylation. These findings show that basal and agonist-stimulated eNOS activity are regulated by different mechanisms. The loss of basal eNOS activity with time in the organ bath and wortmannin treatment and the concomitant decline in Akt phosphorylation point to involvement of the PI3K/Akt pathway and tyrosine phosphorylation, but not of eNOS phosphorylation of at S1177.