To evaluate the effect of topical application of tetracaine on intraocular pressure (IOP) measurement by Tonopen in dogs. Six healthy male Epagneul Bretons (group 1) and six healthy male black Labrador Retrievers (group 2) were examined. IOP was measured in the right eye (OD) prior to (IOP1) and 1 minute following instillation of one drop of topical tetracaine (IOP 2), and the left eye (OS) (control) prior to (IOP 3) and 1 minute following instillation of one drop of isotonic saline solution (IOP 4). Measurements were performed on two occasions: at 8:00 AM and 3:00 PM. For both groups, IOP measurements were higher in the morning than in the afternoon. For group 1, IOP1 mean (SD), IOP2 mean (SD), IOP3 mean (SD) and IOP4 mean (SD) were 14.6 (2.2) mmHg, 11.3 (3.2) mmHg, 14.4 (2.2) mmHg and 13.5 (3.9) mmHg respectively, while in group 2, IOP1 mean (SD), IOP2 mean (SD), IOP3 mean (SD) and IOP4 mean (SD) were 14.2 (3.8) mmHg, 9.5 (3.7) mmHg, 13.5 (2.8) mmHg and 13.0 (3.8) mmHg respectively. For both groups at each time point, IOP 2 values were significantly lower (P<0.007) than IOP 1 values, whereas IOP 3 and 4 values were not significantly different (P>0.27). This study demonstrates that topical application of tetracaine significantly lowers IOP measured by Tonopen due to a possible interaction with melanin. The potential effect of topical anesthetics should be taken in consideration when performing applanation tonometry for clinical, pharmacological and toxicological studies.