A Tono-Pen-1 (TP-1) and a Tono-Pen-2 (TP-2) were calibrated against direct manometry in the eyes of living rabbits to determine the accuracy of the Tono-Pen. Eighteen normal eyes of 10 rabbits were cannulated and connected to a pressure transducer with a chart recorder. Intraocular pressure (IOP) was increased from 5 to 40 mm Hg in 5-mm-Hg increments and from 40 to 80 mm Hg in 10-mm-Hg increments. After each incremental increase, IOP was measured first with a TP-1 and then with a TP-2. Plotting the mean Tono-Pen readings for each eye against the transducer pressure produced the following two regression line formulas: y = -0.613+0.790x (r2 = 0.97) for TP-1, and y = -1.45+0.824x (r2 = 0.97) for TP-2. Neither regression line was statistically different; p = 0.2 for the two slopes, and p = 0.24 for the y-axis intersection. The TP-1 and TP-2 can be used to rapidly and accurately measure the IOP in normal rabbit eyes. Tono-Pen readings do, however, significantly underestimate pressures in the range of 5-80 mm Hg. A table with 95% confidence intervals for corrected IOP readings obtained with the TP-1 or -2 is provided.