INTRODUCTION: There has been increased attention on the potential impact of the preservative benzalkonium chloride (BAK) on the ocular surface. This study compared the ocular surface tolerability of once-daily bimatoprost 0.01% and latanoprost 0.005% (both preserved with 0.02% BAK), and travoprost 0.004% preserved with sofZia™. METHODS: A randomized, multicenter (15 sites), investigator-masked study enrolled patients with open-angle glaucoma or ocular hypertension who had received latanoprost monotherapy for at least 1 month. Patients were randomized to oncedaily bimatoprost (n = 56), travoprost (n = 53), or latanoprost (n = 55) monotherapy for 3 months. Follow-up visits were at weeks 1, 4, and 12. The primary outcome measure was physician-graded conjunctival hyperemia (scale 0 to 3) at week 12. Secondary outcomes included corneal staining (scale 0 to 3) and tear break-up time (TBUT). RESULTS: There were no significant differences in mean (standard deviation [SD]) outcome measures including conjunctival hyperemia (bimatoprost: 0.48 [0.52], travoprost: 0.49 [0.52], latanoprost: 0.51 [0.54]), corneal staining (bimatoprost: 0.31 [0.49], travoprost: 0.25 [0.46], latanoprost: 0.24 [0.45]), or TBUT (bimatoprost: 9.7 s [6.1], travoprost: 9.5 s [5.8], latanoprost: 9.8 s [5.0]) among subjects at latanoprost-treated baseline (P ≥ 0.664). At week 12, there were no significant differences in conjunctival hyperemia (bimatoprost: 0.42 [0.48], travoprost: 0.46 [0.44], latanoprost: 0.44 [0.57]), corneal staining (bimatoprost: 0.31 [0.45], travoprost: 0.32 [0.48], latanoprost: 0.22 [0.30]), or TBUT (bimatoprost: 9.7 s [5.7], travoprost 9.7 s [5.0], latanoprost: 9.3 s [4.0]) among the treatment groups (P ≥ 0.379). At week 1, there was a statistically significant among-group difference in mean change from baseline in hyperemia (+0.04, bimatoprost; +0.20, travoprost; 0.00, latanoprost; P = 0.018). There were no statistically significant among-group differences in mean corneal staining, mean TBUT, or change from baseline at any visit. CONCLUSIONS: Despite preservative differences, there were no significant differences in objective clinical measures of ocular surface tolerability after 3 months of treatment with bimatoprost (with 0.02% BAK), travoprost (with sofZia), and latanoprost (with 0.02% BAK).