Recent evidence suggests a key role of bacterioplankton in shaping the composition of the dissolved organic matter (DOM) pool in aquatic systems, not only through consumption but also through production of specific compounds, but the latter process is still not well understood. We used a bioassay approach to assess the patterns in bacterial production and consumption of five fluorescent DOM pools in seven lakes and two streams in Southeastern Québec, Canada, and the links these patterns may have with key aspects of bacterial metabolism, DOM origin and nutrients availability. Total dissolved organic C declined by 3-15% during these incubations, whereas the specific DOM pools had very different dynamics: Two humic-like fractions accumulated in all incubations, with rates of production increasing as a function of bacterial growth efficiency, which itself increased with phosphorus concentrations. In contrast, two protein-like fractions and a third humic-like fraction either increased or declined over the course of the experiments. The net production or consumption of these pools appeared to be a function of the contribution of terrestrial C to bulk DOM (derived from δ(13) C of the DOM) and of total bacterial activity. Our results suggest that lake bacterioplankton play a dual role in DOM dynamics, as consumers and also producers, and that the interplay between DOM origin and nutrient availability appears to determine the net outcome of bacterial DOM processing, thus influencing the bulk DOM composition and its fate in these aquatic systems. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.