This study proposes to evaluate the level of accuracy of intraocular pressure (IOP) measurements of a second generation rebound tonometer (IOPen, taking as references the Goldmann Applanation Tonometer (GAT) and the iCare rebound tonometer. The right eyes of 101 consecutive clinical patients were assessed with the three tonometers. The IOPen and iCare measurements were taken by two different optometrists and the GAT by an ophthalmologist. In this study, statistically significant differences were found when comparing the IOPen tonometer with the other two tonometers (p < 0.001). The IOPen underestimated the IOP value when compared to the GAT and the iCare (mean differences were 2.94 +/- 4.65 mmHg and 3.20 +/- 4.72 mmHg (mean +/- S.D.), respectively). The frequency distribution of differences demonstrated that in more than 55% of measurements the IOP readings differed by more than 3 mmHg between the IOPen and the GAT. Based on the present population study, these results suggest that IOPen measurements should be interpreted with caution.