Much is known about the spatial and temporal distribution of macro-organisms and their activity rates. However, little is known about the spatial variability of microbial communities in lakes. In this study, we examined the spatial variability in both bacterial community composition and activity rates in Toolik Lake, Alaska. Community composition was characterized using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified rDNA, and activity rates were measured by uptake rates of 14C-leucine. Results from a single survey in 2007 indicate that activity rates and community composition of Toolik Lake bacterial communities vary spatially throughout the water column and horizontally across a water layer. In 2008, seasonal changes showed that early and mid-season samplings of Toolik Lake communities exhibited different spatial patterns of activity and composition than the late season sampling. In the early and mid-season (June and July), spatial variability in activity rates and community composition was high throughout the water column, and across the surface waters. A large storm event in the late season homogenized the activity rates and community composition of surface waters and shallow hypolimnion sites. During the storm event, only the deepest sites maintained large differences compared to other sites, demonstrating the importance of mixing events for controlling the spatial pattern of activity rates and community composition in lake ecosystems. In addition, we found a seasonal succession of the microbial community throughout the lake. Our results suggest that microbial communities can vary substantially within a single lake, even within a distinct water layer. This degree of spatial structure can change predictably with mixing events and over the course of a season.