The change in peak III/I ratio of pyrene monomer fluorescence, a measure of the hydrophobicity of pyrene's environment, was used to examine the aggregation behaviour of chitosan in aqueous solution. The III/I ratio of 0.60 at chitosan concentrations below 0.1 mg/ml was very similar to that observed in water and other polar media. Increasing the chitosan concentration above 1.0 mg/ml, however, significantly increased the III/I ratio. The III/I ratio was 0.73 in chitosan-70 K and 1.12 in chitosan-750 K when the concentration was increased to 5.0 mg/ml. The values of 0.73 and 1.12 correspond to pyrene being localized in an environment similar to methylene chloride and isopropyl alcohol, respectively. The increased hydrophobicity suggests that chitosan chains were self-associated by intermolecular hydrophobic interactions. No difference in the III/I ratio was found between solutions made in 0.1 M acetic acid and 0.1 M hydrochloric acid. Changes in the ionic strength by addition of sodium chloride, however, did significantly affect the aggregation behavior.