BACKGROUND Application of timolol maleate (TM) in a conventional dosage form (solution) into the eye results in almost 80% of the instilled dose being lost either through spillage or due to drainage into the nasolacrimal duct. Later results in systemic side-effects especially in patients suffering from heart diseases or bronchial asthma thus limiting the usefulness of TM for the control of glaucoma. Earlier we had reported on the development of a mucoadhesive coated niosomal system for TM (TM REV(bio)) containing 0.25% TM. Presently we establish and report the pharmacokinetic and pharmacodynamic superiority of the developed ocular formulation of TM. METHODS Aqueous humor concentration of TM in male albino rabbits, after instillation of one drop of TM solution (TMS) or TMREV(bio) was measured using the microdialysis method. RESULTS Peak concentration of drug in aqueous humor from TMREV(bio) (12.46 microg/ml achieved at 60 min) was almost 1.7 times that of the control drug solution (TMS, 0.25%; 7.2 microg/ml). An important observation was that the high drug concentrations achieved upon TMREV(bio) administration were maintained for up to 2 h. AUC for TMREV(bio) formulation was 2.34 times that of the TMS. CONCLUSIONS This study confirms a sustained and controlled effect of the developed formulation.