NOS3-mediated NO regulates endothelial and kidney epithelial function. Lysine acetylation is a dynamic post-translational modification influencing cell signaling. Our lab and others showed NOS3 is regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites of NOS3 and investigated lysines in the NOS3 reductase domain, K607, K610, K727, and K731. We hypothesized that deacetylation of one or more of these lysines on NOS3 would impact NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation by HPLC in the media over 48 hours. Mutation of all four lysines or lysines K607 and K610 dramatically increased NO production whereas K727 and K731 did not change NO production compared to wildtype (pmol NO2 /mg protein, WT: 2271 ± 729.3; K607/610/727/731R: 15176 ± 2759, p=0.046 vs WT; K607/610R: 9431 ± 1772, p=0.0019 vs WT; K727/731R: 3523 ± 805.8, p=0.918). We further investigated single mutation of K607 and K610. We found K610R (10564 ± 1883 pmol NO2 /mg protein, p=0.002 vs WT), but not K607R (1589 ± 412.0 pmol NO2 /mg protein, p>0.99) to have increased NO production compared to WT (1547 ± 180.8 pmol NO2 /mg protein). We then hypothesized that acetylation of K610 on NOS3 would decrease NO production compared to deacetylation of K610. We mutated K610 to glutamine mimicking acetylation of this lysine. Contrary to what we hypothesized, we found K610Q to produce greater NO than both WT and K610R (pmol NO2 /mg protein, WT: 2196 ± 606.1, K610Q: 30212 ± 880.2, p<0.0001 WT vs K610Q; K610R:13389 ± 1086, p<0.0001 K610R vs K610Q). Previous reports show that amino acids 605-612 coordinate regulatory phosphorylation sites of NOS3. We therefore investigated NOS3 K610R and K610Q relative to WT at key phosphorylation regulatory sites by western blot. We found K610R and K610Q to have reduced phosphorylation at T495 (RDU; WT: 1.00 ± 0.252; K610R: 0.820 ± 0.251, p=0.0257; K610Q: 0.711 ± 0.205, p=0.0046), increased phosphorylation at S1177 (WT: 1.00 ± 0.125; K610R: 1.58 ± 0.2921, p=0.038; K610Q: 1.93 ± 0.358, p=0.0082), and no change in phosphorylation at S633 (WT: 1.00 ± 0.054; K610R: 0.885 ± 0.0536, p=0.126; K610Q: 0.955 ± 0.0779, p=0.319). Additionally, K610R but not K610Q was found to have increased S615 phosphorylation (WT: 1.00 ± 0.112; K610R: 2.01 ± 0.259, p=0.0003; K610Q: 1.07 ± 0.112, p=0.682). These data suggest acetylation of K610 would significantly increase NO production and regulate NOS3 phosphorylation sites in endothelial cells and/or kidney epithelial cells. We propose that acetylation/deacetylation of K610 would have dramatic consequences for blood pressure regulation as well as water and electrolyte homeostasis.