Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([ 14 C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and Actinobacteria. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.Estuarine waters contain strong biological and chemical gradients established by the mixing of freshwater and seawater and modified by autochthonous biological activity. Many of these gradients, including salinity, nutrient concentration, organic matter composition, and bacteriovore community composition, are thought to influence the composition of natural bacterioplankton communities (2, 11). Such changes in environmental conditions, when recreated in mesocosm and microcosm experiments, caused shifts in the phylogenetic composition of bacterioplankton communities (10,19,32,41). It is therefore reasonable to predict that similar shifts will occur in natural freshwater and marine bacterioplankton communities when they encounter estuarine gradients, leading to the development of an estuarine community.Several studies have described estuarine microbial diversity and some have demonstrated how freshwater and marine bacterioplankton communities mix along estuarine gradients (3,4,8,14,21,37), but few reports have provided evidence of a unique estuarine bacterioplankton community. This is partly due to the dynamic nature of estuaries and the difficulty in distinguishing estuarine populations from those that wash in from adjacent environments. Crump et al. (6) identified putative estuarine bacteria associated with particles in the Columbia River estuarine turbidity maximum (ETM) by comparing environmental clone libraries of PCR-amplified 16S ribosomal DNA (rDNA) from the river, the estuary, and the coastal ocean. Similarly, Hollibaugh et al. (14) demonstrated the mixing of bacter...