The objectives of this study were to determine the concentration of itraconazole achieved in corneal tissue and aqueous humour after topical application of a 1% itraconazole ointment: to determine the effect of including dimethyl sulphoxide (DMSO) in the ointment on achievable ocular tissue itraconazole concentrations; and to assess if any gross or histopathologic ocular toxicity results from the topical application of 1% itraconazole with or without the addition of DMSO. The experimental trial consisted of 6 horses considered to have normal eyes. Each horse had one eye treated with 0.3 mL of 1% ultra-micronized itraconazole ointment and the fellow eye with 0.3 mL of 1% itraconazole/ 30% DMSO ointment. The ointment was applied every 6 h for a total of 28 treatments. Both ointments were well tolerated and no gross or histopathologic abnormalities developed during the trial. Corneal tissue and aqueous humour concentrations of itraconazole were determined using high performance liquid chromatography. Corneal tissue concentration averaged 1.1 (+/- 0.4) micrograms/g in horses treated with the 1% ultramicronized itraconazole ointment and 7.9 (+/- 3.3) micrograms/g for those treated with the 1% itraconazole/30% DMSO ointment: there was a statistically significant difference between ointments (P = 0.005) No itraconazole could be detected in the aqueous humour in either treatment group.