The molar absorption coefficient of pyrene aggregates, epsilon(E0), was determined for a series of pyrene-labeled poly(N,N-dimethylacrylamide)s (Py-PDMA) having different pyrene contents. Aqueous solutions of Py-PDMA having pyrene contents ranging from 263 to 645 mu mol of pyrene per gram of polymer were studied by UV-vis absorbance and time-resolved fluorescence spectroscopy. The global analysis of the monomer and excimer fluorescence decays with the fluorescence blob model yielded the fractions of the overall absorption contributed by all the pyrene species present in solution. The combined knowledge of the fractions obtained from the global analysis of the time-resolved fluorescence decays, the total absorption of the Py-PDMA solution obtained from UV-vis spectroscopy, and the total pyrene concentration in the solution obtained from the known pyrene content of each Py-PDMA sample led to the determination of the molar absorption coefficient of pyrene aggregates. Regardless of the pyrene content of the Py-PDMA samples and hence the level of association of the pyrene pendants in solution, all Py-PDMA samples yielded similar epsilon(E0) values over the range of wavelengths studied, namely, from 325 to 350 nm. The averaged epsilon(E0) was found to be red-shifted relative to unassociated pyrenes by 3 nm as well as having a value at the 0-0 peak of 21 000 M-1.cm(-1) reduced from 34 700 M-1.cm(-1) for unassociated pyrenes. The determination of epsilon(E0) enabled the first determination of the absolute fraction of associated pyrenes for aqueous solutions of a series of pyrene-labeled water-soluble polymers. The procedure outlined in this study is applicable to any pyrene-labeled water-soluble polymer and provides a new means to study quantitatively the effect of the hydrophilic-to-lipophilic balance on the hydrophobic associations generated by hydrophobically modified water-soluble polymers. As an application, the average number of pyrenes involved in a pyrene aggregate generated by Py-PDMA in water is determined.