<p>Hydration of materials affects their antifouling properties, and polyethylene glycol (PEG) is highly hydrated and biocompatible. PEGylated dendrimers have been studied as drug carriers that passively accumulate in tumors owing to their enhanced permeability and retention effect. In this study, we synthesized various PEGylated dendrimers with different PEG molecular weights, bound numbers, and dendrimer generations, and analyzed their hydration properties using differential scanning calorimetry and Fourier transform infrared spectroscopy. Intermediate water is defined as water molecules loosely bound to a material, the amount of which has been reported to be a useful index of bioinert properties. The amount of intermediate water was estimated from enthalpy change in melting peaks below 0 °C, considering a PEG-water eutectic system. Our results indicate that intermediate water was richer in PEGylated dendrimers with longer and more PEG chains. These PEGylated dendrimers, with longer and more PEG chains, also showed enhanced tumor accumulation after intravenous injection. The tumor-to-liver ratio correlated with the intermediate water content in the PEGylated dendrimers. This suggests that hydration is an important factor influencing dendrimer biodistribution, which is a possible criterion in designing nanocarriers for drug delivery systems.</p>