Ocular hypertension in glaucoma arises from increased drainage resistance for aqueous humor in the conventional outflow pathway, which includes the trabecular meshwork (TM) and Schlemm’s canal (SC). Unfortunately, the cellular mechanisms responsible for resistance generation are largely unknown. Our previous funding cycles have demonstrated that nitric oxide (NO) is a key regulator of outflow resistance and intraocular pressure (IOP). Moreover, we have shown that shear stress stimulates NO production by SC endothelial cells, like vascular endothelia. Further, the magnitude of shear stress acting on SC cells depends on IOP, due to pressure-induced narrowing of the SC lumen. As NO is known to decrease outflow resistance, shear-induced NO production may act as a “fast” homeostatic signal to oppose the source of IOP elevation and help to maintain IOP in a narrow range. This “fast” homeostasis is sensed by SC shear stress and operates over time scales of seconds to minutes, and contrasts with the “slow” IOP homeostasis that is sensed by TM stretch that stimulates extracellular matrix (ECM) remodeling over several days. These “fast” and “slow” mechanisms are complementary because they allow the outflow pathway to sense and respond to perturbations in outflow function over a range of temporal scales, from acute outflow obstruction to chronic remodeling of ECM. Our recent data provide further insight into the homeostatic role of NO and how NO maintains the health and function of the conventional outflow pathway. For example, our data show that NO production by SC cells is amplified by pulsatile shear stress, which arises due to the ocular pulse and results in an immediate pulsation-induced decrease in outflow resistance. We also show that NO contributes to the clearance of particulate matter, such as pigment and cell debris, that would naturally accumulate in the juxtacanalicular TM. Thirdly, our modelling studies suggest that elevated TM stiffness (as occurs in primary open angle glaucoma; POAG) eliminates the “fast” IOP homeostasis by suppressing pulsation-induced shear stress in SC. Consequently, this desensitization allows debris to accumulate unchecked in the TM, leading to eventual outflow dysfunction and IOP elevation. Taken together, our central hypothesis is that NO has two critical roles in maintaining IOP homeostasis over short time scales: (i) as a key signaling molecule in a mechanosensitive feedback loop potentiated by pulsatile shear stress in the SC lumen, and (ii) as a modulator of inner wall permeability and TM contractility to flush cell debris/pigment from the juxtacanalicular TM. We test our hypothesis with three Specific Aims (SAs). SA1: To determine how the ocular pulse modulates outflow facility through NO signaling. SA2: To determine how NO contributes to IOP homeostasis in response to particulate load in the TM. SA3: To determine how NO regulates inner wall permeability, enabling particulate clearance from the TM. Impact: Outcom...