The objective of this study was to maximize the in vitro transcorneal release, the intraocular pressure (IOP) lowering effect and the duration of action, of the Latanoprost acid (LAT) ophthalmic gels. The in vitro transcorneal release of LAT from a first set of gel formulations containing different concentrations of Azone (as enhancer) with a fixed concentration of C-974® (as mucoadhesive) was studied. The formulation that showed the greatest permeability at the lowest Azone concentration was selected for the preparation of a second set of ocular gels containing different C-974® concentrations. Their in vitro permeabilities were evaluated, and the C-974® concentration yielding the greatest in vitro permeability was chosen. The in vivo IOPlowering efficacy study for the scaled-up formulations from both sets of the test formulations was performed using a Tono-pen Avia® tonometer in rabbits for 4 consecutive days. To determine the duration of action, the most effective formulations were used for a single-dose study, and the IOP was measured at predetermined intervals until the IOP base-line was reestablished. The majority of the tested formulations showed significant but varied augmentations in both the in vitro and in vivo permeability results. The formulations GAZ-4 and GC-4 showed the greatest IOP lowering effects, i.e., 7.8±1.8 mmHg and 6.5±2.1 mm Hg, respectively. It is particularly noteworthy that for both formulations the IOP lowering effect continued for 24 hours. Their duration of action in the single-dose study were 47±2.25 hours and 48±1.5 hours, respectively. It was concluded that the in vitro release, onset, magnitude and duration of action of the LAT gels were increased and extended for up to 2 days for the two gel formulations.