Dissolved organic matter (DOM) composition depends on the sources and degree of processing. We investigated DOM bioavailability and sources in a prairie stream.  Bioavailability was calculated as the percent change in DOC over month-long lab incubations.  DOM was characterized by fluorescence component composition (excitation-emission matrixes fit to a previously published parallel factor model), to determine relationships among DOM source, bioavailability, and composition.  Mean DOC bioavailability was 36%, but exhibited spatial and temporal variation, ranging from 0–70% in surface waters.  Fluorescence index, a proposed measure of relevant contribution of microbial and terrestrial sources of DOM, was not significantly correlated with bioavailability, and exhibited a 0–42% change responses to degradation in leachates.  Stream DOM composition exhibited greater than 90% similarity to groundwater, but similarity decreased downstream.  Stream DOM was most similar to prairie soil, senescent grass, and algal leachates, and similarity to stream water increased following leachate degradation.  DOM fluorescence is a powerful tool for the study of DOM in the natural environment and may help resolve the main sources of DOM and spatial and temporal variability in contributions to DOM in streams.