Utilization of 95 carbon sources by bacteria in 2 Canadian Shield lakes and 2 Texan reservoirs was determined using Biolog-GN microtiter plates. Triplicate plates were inoculated and incubated for 5 d, during which color development was monitored twice daily by optical density (OD595). Optical densities in plate wells containing carbon substrates were corrected for blank absorbance at each measurement time. Then, data on optical densities of all substrate wells were selected from a single measurement time, to construct a community-level physiological profile. This measurement time was chosen so that plates would be compared at similar levels of color development. This data selection also generally maximized correlations of substrate utilization patterns determined from replicate plates. Multivariate analyses (ordinations) of the community-level physiological profiles identified several amino acids, carboxylic acids, and carbohydrates associated with seasonal patterns of substrate utilization. Multivariate analyses further revealed patterns in substrate utilization common to all 4 lakes: strong relative responses to amino and carboxylic acids in cool seasons and strong relative responses to carbohydrates in warm seasons. These patterns may be driven by seasonal events among phytoplankton that influence carbohydrate supply, and appear to be dampened in an oligotrophic lake, which does not support high algal abundance.