PURPOSE: To analyze the effect of using one reading, the mean of two readings (from the same eye), or the mean of four readings (two from each eye) on the heritability estimates of intraocular pressure (IOP). This was a cohort study in which 344 pairs of twins, 163 monozygotic (MZ) and 181 dizygotic (DZ), were enrolled. METHODS: IOP was measured using three tonometers: the gold standard Goldmann applanation tonometer (GAT), the Ocular Response Analyzer (ORA; Reichert Buffalo, NY), and the Dynamic Contour Tonometer (DCT, Pascal; Swiss Microtechnology AG, Port, Switzerland). The main outcome measure was the heritability of IOP correlated with the number of measurements. RESULTS: The mean IOPs of all four readings with the three tonometers were: 14.1 +/- 2.9 mm Hg for GAT, 15.9 +/- 3.2 mm Hg for ORA, and 16.9 +/- 2.7 mm Hg for DCT. As the number of readings increased, the calculated heritability (h(2)) of IOP measured using the GAT readings increased from 0.56 for one reading (95% confidence interval [CI], 0.44-0.65) to 0.58 for the mean of two readings (95% CI, 0.46-0.67) to 0.64 for the mean of all four readings (two right and two left; 95% CI, 0.55-0.72). Similar results were seen with the other two instruments. CONCLUSIONS: The results demonstrated that the use of the mean of several readings from both eyes reduced measurement error, yielding a higher heritability estimate. The higher heritability would increase the power to detect linkage in a genome-wide analysis.