The aim of this study was to prepare bioadhesive sulfacetamide sodium (SA) microspheres to increase their residence time on the Ocular Surface and to enhance their treatment efficacy on Ocular keratitis. Microspheres were fabricated by spray drying method using mixture of polymers such as pectin, polycarbophil and hydroxypropylmethyl Cellulose (HPMC) at different ratios. The particle size and distribution, morphological characteristics, thermal behavior, encapsulation efficiency, mucoadhesion and in vitro drug release studies on formulations have been investigated. After optimisation studies, SA-loaded polycarbophil microsphere formulation with polymer:drug ratio of 2:1 Was found to be the most Suitable for Ocular application and used in in vivo studies. In vivo studies were carried Out on New Zealand male rabbit eyes with keratitis caused by Pseudomonas aeruginosa and Staphylococcus aureus. Sterile microsphere Suspension in light mineral oil was applied to infected eyes twice a day. Plain SA Suspension Was used as a positive control, On 3rd and 6th days of the antimicrobial therapy, the eyes were examined in respect to clinical signs of infection (blepharitis, conjunctivitis, iritis, corneal oedema and corneal infiltrates) which are the main symptoms of bacterial keratitis and then cornea samples were counted microbiologically. The rabbit eyes treated with microspheres demonstrated significantly lower clinical scores than those treated with SA alone. A significant decrease in the number of viable bacteria in eyes treated with microspheres was observed in both infection models when compared to those treated with SA alone. In conclusion, in vitro and in vivo studies showed that SA-loaded microspheres were proven to be highly effective in the treatment of ocular keratitis. (C) 2009 Elsevier B.V. All rights reserved.