OBJECT To assess the results from using a commercially available phenol red thread (PRT) test with the eyes being kept open (as recommended by the company) vs a closed eye protocol (as used by some investigators). METHODS A single PRT (Zone-Quick) test was carried out on 97 young adults (average age 21 years, range: 17-36) for 15 s with either the eyes open or closed. The right eye was assessed with 52 subjects undertaking the open eye test first, with the test being repeated 1 h later in the same indoor environment (temperature 21 degrees C and 40% humidity). RESULTS The mean ( +/- S.D.) PRT value for the open eye protocol was marginally higher (at 20.5 +/- 7.0 mm) than for the closed eye (19.7 +/- 5.9 mm), but the overall outcome from the two protocols could range from being 18 mm greater in the open eye to 15 mm greater in the closed eye. Overall, the difference was not statistically different, neither were any substantial differences noted according to age, gender, iris colour, prior contact lens wear or according to which protocol was used first (p >or= 0.2). CONCLUSION These studies indicate that there can be a small difference in single PRT test data according to whether the eyes are kept open or closed, and it would be useful if the protocol used was clearly stated.