The Pluronic polyol F127, PEO99PPO69PEO99 (PEO and PPO being poly(ethylene oxide) and poly(propylene oxide), respectively) has the potential to be used as an effective separation medium in capillary electrophoresis (CE) for the separation of biomacromolecules such as DNA fragments and proteins. Static light scattering (SLS), dynamic light scattering (DLS), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) were used to characterize the solution properties and the microstructures of F127 in the same electrophoresis buffer used in CE. In the dilute solution region, F127 in CE buffer forms micelles similar to that in water. At high solution concentrations, micelles tend to pack into some crystalline forms with relatively well-structured PPO centers. By using a combination of SANS and SAXS results, we are able to conclusively determine the gellike structure to have a face-centered cubic lattice. The effects of solvent, polymer concentration, temperature, and sample preparation procedure on the gel structure were studied. In the gellike region the aggregation number and also the micellar size are not sensitive to both the concentration change and the temperature change. The results of DNA electrophoretic migration in F127 gels also support these findings.