Recurring seasonal patterns of microbial distribution and abundance in three third-order temperate streams within the southeast Pennsylvania Piedmont were observed over 4 years. Populations associated with streambed sediments and rocks (epilithon) were identified using terminal restriction length polymorphism (tRFLP) and sequencing of 16S rRNA genes selectively amplified with primers for the bacterial domain. Analyses of the relative magnitudes of tRFLP peak areas by using nonmetric multidimensional scaling resolved clear seasonal trends in epilithic and sediment populations. Oscillations between two dominant groups of epilithic genotypes, explaining 86% of the seasonal variation in the data set, were correlated with temperature and dissolved organic carbon. Sequences affiliated with epilithic phototrophs (cyanobacteria and diatom chloroplasts), a Rhodoferax sp., and a Bacillus species clustered in the summer, whereas sequences most closely related to "Betaproteobacteria" (putative Burkholderia sp.), and a putative cyanobacterium clustered in the fall/spring. The sediment genotypes also clustered into two groups, and these explained 85% of seasonal variation but correlated only with temperature. A summer tRFLP pattern was characterized by prevalence of "Betaproteobacteria," "Gammaproteobacteria," and a Bacillus sp., whereas the winter/spring pattern was characterized by phylotypes most closely related to "Firmicutes," "Gammaproteobacteria," and "Nitrospirae." A close association between these headwater streams and their watersheds was suggested by the recovery of sequences related to microbial populations provisionally attributed to not only freshwaters but also terrestrial habitats.Headwater streams are a primary link between aquatic and terrestrial ecosystems, transforming and exporting terrestrially derived materials and also contributing to export through autochthonous primary production. Complex communities of macro-and microbiota distributed among different stream habitats primarily mediate these transformations. The trophic structures of stream macrobiota and phototrophic eukaryotic microbiota have been intensively studied as contributors to production and energy flow in lotic systems (26). In contrast, although microorganisms exert significant, if not major, control on system chemistry and the processing of terrestrial inputs, there is little understanding of differences in lotic microbial population structure among biomes, population similarity among streams within a biome, or changes in structure associated with seasonal oscillations in chemical and physical parameters. This information is essential for developing a more complete description of the trophic structure and biogeochemistry of streams, one that more fully incorporates their relationship to the proximal terrestrial system (11,12,16,47,48).Microbial communities in the temperate deciduous forest biome are exposed to seasonal changes in their chemical and physical environments. Some seasonal changes are constrained, such as variation in light a...