A case study of hyporheic research in a northern Michigan river is presented to illustrate some potential effects of subsurface hydrology on microbial ecology. Hydrologic flow between a downwelling and upwelling zone beneath a riffle-pool sequence promoted hyporheic-groundwater interaction and differentiated regions of increased bacterial activity, production, turnover time, and responsiveness to dissolved organic carbon enrichment. Large gaps in knowledge exist on the structure and function of fungal, bacterial, microalgal, protozoan, and micrometazoan assemblages within hyporheic zones. This study, combined with available literature on surface and groundwater microbial assemblages, generated several hypotheses for future research. Hydrological methods, when coupled with biochemical measurements, micro-techniques, and descriptions of hyporheic physicochemical and biological gradients, can potentially contribute to a greater understanding of the role of hyporheic microbial communities in the structure and function of lotic ecosystems.