We report experiments based upon fluorescence resonance energy transfer (FRET) measurements designed to examine mixing at the molecular level of the components of a waterborne 2K polyurethane (WB2KPU) formulation. The system consists of an acrylic polyol latex (M-n = 4200 g/mol, D = 2, T-g approximate to 15 degrees C) with a uniform hydrodynamic diameter (d(h)) approximate to 120 nm plus a water-dispersible polyisocyanate (hmPIC, Basonat HW1000 from BASF). We prepared components labeled with phenanthrene (Phen) as the donor dye or with a dimethylaminobenzophenone (Nben) as the acceptor dye. Dynamic light scattering was used to monitor the size and size distribution of the components in the dispersed phase in solution. This signal was dominated by the polyol nanoparticles, which were much larger than the tiny droplets formed by the hmPIC in water. Experiments were carried out at a mole ratio of NCO/polyol-OH of 1.3. We found that the particle size and narrow size distribution remained unchanged up to 22 h after mixing the polyol with the PIC. FRET experiments were carried out on samples in the dispersed state as well as on films formed from these dispersions. Films formed from a 1:1 mixture of (polyol-Phen + polyol-Nben) showed relatively little energy transfer (Phi(ET) = 0.19) even after several hours aging at ambient temperature, indicating that little polymer diffusion occurred in these low-molecular-weight latex films. In contrast, films formed from mixtures of (polyol-Phen + polyol-Nben + hmPIC) showed more extensive energy transfer (ET) (Phi(ET) = 0.51), indicating essentially complete mixing at the molecular level of the polymer molecules in the presence of hmPIC. The key conclusion is that hmPIC is a reactive plasticizer that promotes diffusion in this system on a much faster scale than the cross-linking reaction. This result is confirmed by experiments that examined mixtures of (hmPIC-Phen + polyol-Nben), which also showed essentially molecular scale mixing between these two different components. In this later system, aging at room temperature led to a small decrease in Phi(ET) over time that was more prominent for films aged at high humidity (75%) than at lower humidity (45%). This result suggests that hydrolysis of NCO groups in the film, leading to polyurea formation, promotes local phase separation accompanied by a net increase in the average separation of Phen and Nben groups in the film.