Aqueous humor (AH) produced by the ciliary body exits the eye through the Trabecular Meshwork (TM) and Schlemm's canal. Impairment of AH outflow produces ocular hypertension that can result in glaucoma development. Contractile state, volume and cytoskeletal architecture of TM cells are important determinants of AH outflow. We found that AH outflow can be regulated by activation/blockade of TM ion channels involved in volume regulation, cell contractility and calcium homeostasis. In ocular anterior segments cell swelling decreases outflow while cell shrinkage produces the opposite effect. Cell swelling activates chloride (Clswell) and K⁺ channels (BKCa) to restore resting TM cell volume. Blockade of these channels impairs outflow facility recovery after hypotonic cell swelling, while other chloride channels such as ClC-2 are not involved. TM cytoskeleton and cellular contractility are also important regulators of outflow facility. Compounds that destabilize actin cytoskeleton (i.e. latrunculins) produce large increases in AH outflow. BKCa channels activation promotes cell and tissue relaxation in response to different vasodilators, increasing outflow facility. In contrast, many compounds present in the AH signal through Gq/11-protein coupled receptors, activating receptor and/or store-operated Ca²⁺ entry after Ca²⁺ release from intracellular stores, likely involving TRPC channels. Traction microscopy shows that endothelin and bradykinin induce actomyosin-dependent cell contraction, decreasing AH outflow. Involvement of TRPC channels in these effects is currently under study. In summary, a balance between different regulatory mechanisms is necessary for AH outflow pathway function. Alteration of this delicate equilibrium by different insults leads to tissue malfunction and development of ocular hypertension.