Purpose: To evaluate the effect of a latanoprost/timolol fixed combination (LTFC) versus a latanoprost and timolol gel-forming solution unfixed combination (LTuFC) on daytime intraocular pressure (IOP) levels and fluctuations. Methods: This was an 8-week, randomized, parallel-group study. Ninety eyes of 90 patients diagnosed with primary open-angle glaucoma or ocular hypertension that were insufficiently controlled with latanoprost monotherapy were enrolled. Randomized patients received either a single evening dose of LTFC or unfixed combination of latanoprost administered once daily in the evening and timolol gel-forming solution administered once daily in the morning without a washout period. IOP measurements were taken at 8 AM, 11AM, 2PM, and 5 PM. The mean IOP, daytime IOP measurements, and fluctuation in IOP were assessed at weeks 4 and 8. Results: The mean IOP reduction from baseline to each visit was significant in both groups (P < 0.01). There was a significant difference in the mean IOP between groups at week 4 (P = 0.0021). At week 8, the mean IOP reduction was 3.2 +/- 2.1 and 5.7 +/- 3.2mm Hg in LTFC and LTuFC groups, respectively, and the difference was significant (P = 0.001). A decrease in the daytime IOP fluctuation was observed in both groups over time, but the decrease in each group was not significant. At week 8, the mean IOP measurements at 8 AM, 11AM, 2PM, and 5 PM in LTuFC group were significantly lower than in the LTFC group (P = 0.024, 0.0001, 0.0008, and 0.0011, respectively). Conclusions: The concomitant use of latanoprost and timolol gelforming solution leads to a larger additional IOP reduction and lower daytime IOP levels as compared with the fixed combination.