Sorption coefficients for pyrene on dissolved humic substances and on poly(acrylic acid) esters as well-defined model polymers were determined using solid-phase microextraction (SPME) and the fluorescence quenching technique (FQT). The results of both analytical methods were compared and theoretically evaluated, which led to the conclusion that the sorption coefficients measured by SPME and HIT are inevitably different: SPME measures activity-based and FIT concentration-based sorption coefficients. The environmental relevance of the two types of sorption coefficients is discussed. HIT is inappropriate to measure sorption coefficients for pyrene with the synthetic sorbents. Inspection of the vibrational structure of the fluorescence spectra of those solutions indicates a highly hydrophobic microenvironment of pyrene. This can be explained by an intra- or intermolecular agglomeration of hydrophobic moieties forming a favorable host for hydrophobic solutes.