Two N,O-[N,N-diethylaminomethyl(diethyldimethylene ammonium) n ]methyl chitosans, coded N+-ChS-2 (degree of substitution, DS = 40%; n = 1.6) and N + -ChS-4 (DS = 132%; n = 2.5), were compared with N-trimethyl chitosan (TMC) for their ability to enhance the permeability of dexamethasone (DMS), a transcellular transport route marker, and fluorescein sodium (NaFlu), a paracellular route marker, across an excised rabbit cornea. The effects on DMS permeability were similar: the enhancement ratio (ER) ranged between 1.70 and 1.84, but the action of N + - ChS-4 was quicker. The ER values for NaFlu were 5.99, 3.31 and 8.52 for TMC, N + -ChS-2, and N + -ChS-4, respectively. The ability of N + -ChS-4, the more effective enhancer, to promote intraocular absorption was confirmed in vivo by administering ophthalmic drops to rabbits. The AUC and C max in the aqueous were increased 1.65 and 1.90 times, respectively, for DMS, and 4.56 and 4.43, respectively, for NaFlu. The paracellular transport of NaFlu was enhanced more than the transcellular transport of DMS; however, the maximum enhanced permeability of NaFlu (1.69 ± 0.25 10 - 6 cm s - 1 ) remained lower than the control permeability of DMS (4.38 ± 0.88 10 - 6 cm s - 1 ).