OBJECTIVE In living cynomolgus monkey eyes, the authors compared manometrically set and measured intraocular pressure (IOP) with simultaneous IOP readings obtained with the Tono-Pen (TP), a handheld applanation tonometer based on the Mackay-Marg principle. METHODS In three pentobarbital-anesthetized cynomolgus monkeys, IOP was set and measured manometrically after anterior chamber cannulation through the peripheral cornea with a 26-gauge needle connected to a vertically adjustable reservoir and a pressure transducer. Intraocular pressure was raised in approximately 5 mm Hg steps from 5 mm Hg to 60 mm Hg and then lowered in 5 mm Hg steps to 5 mm Hg, with TP measurements taken at each increment and decrement in open and stopcock modes. RESULTS Linear regression analysis of TP on manometric readings for grouped data from all six eyes, with each data point representing the average of all the TP readings from one eye at each manometric pressure setting, showed a slope 0.692 +/- 0.016 and 0.683 +/- 0.023 (both significantly different from 1; P < 0.001), intercept 1.21 +/- 0.60 and 1.64 +/- 0.82 mm Hg (both significantly different from 0.0, P < 0.05), and correlation coefficient 0.981 and 0.96 in open stopcock and closed stopcock mode, respectively. There were no striking differences when the data were analyzed for individual eyes or animals, for open versus closed stopcock manometry, or for increasing versus decreasing manometric IOP. CONCLUSIONS The TP provides reproducible measurements of IOP in cynomolgus monkeys, with measurement accuracy dependent on the generation of an appropriate calibration curve.