Characterizing dissolved organic matter (DOM) composition remains a major unresolved problem in aquatic ecology and is one of the key impediments to developing a good understanding of DOM production and consumption by heterotrophic bacteria. Fluorescence spectroscopy has been proposed as a promising method for characterizing DOM, but few links have been demonstrated between DOM fluorescence and DOM composition or the processes affected by DOM composition. In 28 southern QueÂbec lakes, tryptophan‐like DOM fluorescence (TFDOM) was found to be a much better descriptor of rates of heterotrophic bacterial metabolism than dissolved organic carbon, describing 52%, 44%, 51%, and 55% of the variability in bacterial production, bacterioplankton respiration, total bacterial carbon consumption, and total plankton community respiration, respectively. In addition, evidence from a series of bacterial regrowth cultures suggests that T‐FDOM represents a product of bacterial activity as well as, to a lesser extent, a bioavailable substrate. Our results instead raise the intriguing possibility that TFDOM concentration reflects a balance between its production and consumption by bacteria. We demonstrate here that fluorescence spectroscopy can be used to identify a highly dynamic fraction of DOM related to bacterial metabolism in lakes.