To develop a protocol to measure the intraocular pressure (IOP) of living mice and to determine the IOP of genetically different mouse strains. Eyes of anesthetifzed animals were cannulated with a very fine fluid-filled glass microneedle. The microneedle was connected to a pressure transducer, and the pressure signal was analyzed with a computer system. Intraocular pressures of male C3H/He iota, C57BL/6 iota, A/iota, and BALB/c iota mice were determined. Differences in IOP were detected between genetically distinct mouse strains maintained in virtually identical environments. C3H/He iota was the strain with the highest average IOP (13.7+/-0.8 mm Hg). This strain average was 1.4 mm Hg higher than that for C57BL/6 iota (12.3+/-0.5 mm Hg; P= 0.14), 4.3 mm Hg higher than that for A/iota (9.4+/-0.5 mm Hg; P< 0.001), and 6 mm Hg higher than that for BALB/c iota (7.7+/-0.5 mm Hg; P< 0.001). The authors have developed an accurate and reliable procedure for measuring intraocular pressure in living mice. This procedure can detect IOP differences between groups of mice that differ by genotype.