The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid precorneal elimination of the drug may be overcome by the use of gel system. The present work was conducted to evaluate the relative bioavailability of ion-activated in situ ophthalmic gel of gatifloxacin by microdialysis. The conventional ophthalmic solution of gatifloxacin was used as reference. The AUC of test group is 3.8-fold vs. the reference group (1.4316 +/- 0.1327 microg.mL(-1).h vs. 0.3756 +/- 0.0380 microg.mL(-1).hr) (P < 0.05), and the C(max) of test group vs. the control group is 3.0-fold (0.3363 +/- 0.0634 microg.mL(-1) vs. 0.1112 +/- 0.0151 microg.mL(-1)) (P < 0.05). The T(max) of test group is longer than that of reference group (2.0 +/- 0.67 hr vs. 0.667 +/- 0.17 hr) (P < 0.1), and K(e) of test group is lower than that of reference group. The developed formulation has a higher bioavailability and longer residence time in aqueous humor than conventional ophthalmic solutions. The developed system is a viable alternative to conventional eye drops.