Changes in selected spectroscopic and chromatographic characteristics of water-soluble organic matter (WSOM) extracted from leaf litter and its ability to bind pyrene were monitored throughout 14 day microbial incubation experiments. To provide additional insight into the microbial transformation of the WSOM, incubation experiments were similarly conducted with controlled-composition mixtures of glucose and dissolved humic substances (HS) that were base extracted from the same leaf litter source. Microbial transformation increased the specific ultraviolet absorbance and number-average molecular weight of residual WSOM while polydispersity values decreased. Fluorescence measurements revealed loss of protein-like fluorescence and enhancement of fulvic- and humic-like fluorescence in residual WSOM. Overall, the incubation results suggest that nonaromatic and smaller sized carbon structures were being degraded while the microbial activity produced humic-like aromatic components in solution. Together, these changes resulted in enhanced pyrene binding by the altered WSOM. Consistent findings resulted from mixtures of glucose and the leaf litter HS. Changes in measured operational descriptors were more pronounced for mixtures containing a higher percentage of glucose, suggesting that utilization of labile constituents may be necessary for formation of unknown structures associated with high pyrene binding capabilities. Simple mass balance, end member mixing models often failed to predict changes in pyrene binding brought about by microbial transformation, suggesting that microbial utilization of labile constituents is not the predominant process governing the enhanced pyrene binding.