Purpose: Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility. Methods: Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGCα and sGCβ isoform expression were determined using Western blot analysis. Results: Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 μL/min per mm Hg [base-line outflow facility] ± 0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGCα and sGCβ subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGC subunit expression was decreased compared with low-passage human TM cells. Conclusions: Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of α and β sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells.