Introduction: Recent research suggests that priapism in sickle cell disease (SCD) is due to dysregulation of penile erection homeostasis including alteration of nitric oxide synthase (NOS) and phosphodiesterase type 5 (PDE5) activities by excessive levels of reactive oxygen species (ROS) released during hemolysis. It is unknown if subacute exposure to hemolysis is sufficient or if chronic reconditioning of erectile tissues is required for perturbation of homeostatic pathways and whether PDE5 inhibitor (PDE5I) treatment can restore erectile homeostasis in the subacute setting. Aims: The aim of this study was to investigate the effects of subacute hemolysis (3-month exposure) on priapism and NO pathway regulation. Methods: Mice underwent bone marrow transplantation with either SCD (BM-SS) or wild-type (WT) bone marrow. BM-SS mice were treated with sildenafil 100mg/kg/day. We measured intracavernous pressure (ICP) measurements with or without cavernous nerve stimulation following bone marrow transplantation to assess for priapism. Main Outcome Measures: ICP and frequency of erections were assessed. Penile tissues were analyzed for NOS, protein kinase G (PKG), PDE5, and ROS activities. Results: BM-SS mice demonstrated a priapism phenotype. PDE5I treatment reduced the frequency of erections in BM-SS mice (1.7±1.1 vs. 5.5±2.8 erections per hour, P<0.05). Penile tissues from BM-SS mice demonstrated decreased NOS, PKG, PDE5 and elevated ROS activities compared with that of control mice. PDE5I treatment increased NOS (11.6±1.3% vs. 7.8±2.3%, P<0.05) and PDE5 (76.3±9.8% vs. 52.3±11.1%, P<0.05) activities and decreased ROS activity (137.8±12.1% vs. 199.1±11.3%, P<0.05) compared with non-PDE5I treated BM-SS mice. PKG activity was increased beyond control levels with PDE5I treatment (158.4±10.3%, P<0.05). Conclusion: Short-term hemolysis is sufficient to establish a priapism phenotype and results in loss of erectile function. PDE5I treatment ameliorates priapism, in part, because of restored NO balance with decreased ROS generation and increased PDE5 activity. Sopko NA, Matsui H, Hannan JL, Berkowitz D, Champion HC, Hsu LL, Musicki B, Burnett AL, and Bivalacqua TJ. Subacute Hemolysis in Sickle Cell Mice Causes Priapism Secondary to NO Imbalance and PDE5 Dysregulation. J Sex Med 2015;12:1878-1885.