The purpose of this study was to determine the effects of three medical treatments, topical oxytetracycline, topical prednisolone acetate, and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. The study design consisted of twelve ophthalmically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (500mg IM bid), topical 1% prednisolone acetate (0.2ml tid), or topical oxytetracycline (0.2ml tid), followed by a 9-day washout period. All topical medications were applied to the left eye and the right eye was treated with a placebo. Tears were collected before the first treatment on day one and the morning following the last treatment on day 5. Tear film proteinase activity was measured using gelatin zymography and measurements of optical density. Statistical analysis of the difference between the treated and untreated eyes and the eyes before and after treatment was performed using mixed effects model for ANOVA. When eyes were compared after treatment, there was no significant difference between treated and placebo eyes for MMP-2 or MMP-9 for any of the treatments. When post-treated eyes were compared to pre-treated eyes, there was a significant decrease in MMP-2 activity in the left eye of horses treated with flunixin meglumine (P=0.0259). There were no differences in MMP-2 and MMP-9 activity for the other treatments. In conclusion, topical 1% prednisolone acetate and topical oxytetracycline did not significantly change MMP-2 or MMP-9 activity in normal equine tear film. Systemic flunixin meglumine had an inhibitory, but questionable, effect on MMP-2 activity in normal equine tear film. This project was funded by Patricia Bonsall Stuart Award for Equine Research.