A simple and reliable method was developed for estimating the hydrophobicity of microenvironments, such as the internal region of aqueous detergent micelles, using a fluorescence probe, pyrene. The partition coefficient of pyrene between hydrophobic environments and water was used as a measure of the hydrophobicity. To validate our method, the partitioning of pyrene between organic solvents of various dielectric constants (n-hexane, 1-octanol, 1-propanol, ethanol, and methanol) and water was measured. This calibration set was then used to estimate the hydrophobicity of the internal region of n-beta-octylglucoside micelles. Combining the spectral changes of pyrene as a function of the n-beta-octylglucoside concentration with a theoretical model of the micellization process, the partitioning of pyrene between the internal region of the micelles and the surrounding water was calculated. The partition coefficient of pyrene was calculated to be about 1.5 x 10(4) based on an assumed aggregation number of 90 for the aqueous n-beta-octylglucoside micelles. Our results demonstrate that the interior of the micelle becomes very hydrophobic once the structure forms.