We examined seasonal and spatial variation in bacterioplankton composition in the Sacramento-San Joaquin River Delta (CA) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Cloned 16S rRNA genes from this system were used for putative identification of taxa dominating the T-RFLP profiles. Both cloning and T-RFLP analysis indicated that Actinobacteria, Verrucomicrobia, Cytophaga-Flavobacterium and Proteobacteria were the most abundant bacterioplankton groups in the Delta. Despite the broad variety of sampled habitats (deep water channels, lakes, marshes, agricultural drains, freshwater and brackish areas), and the spatial and temporal differences in hydrology, temperature and water chemistry among the sampling campaigns, T-RFLP electropherograms from all samples were similar, indicating that the same bacterioplankton phylotypes dominated in the various habitats of the Delta throughout the year. However, principal component analysis (PCA) and partial least-squares regression (PLS) of T-RFLP profiles revealed consistent grouping of samples on a seasonal, but not a spatial, basis. beta-Proteobacteria related to Ralstonia, Actinobacteria related to Microthrix, and beta-Proteobacteria identical to the environmental Clone LD12 had the highest relative abundance in summer/fall T-RFLP profiles and were associated with low river flow, high pH, and a number of optical and chemical characteristics of dissolved organic carbon (DOC) indicative of an increased proportion of phytoplankton-produced organic material as opposed to allochthonous, terrestrially derived organic material. On the other hand, Geobacter-related beta-Proteobacteria showed a relative increase in abundance in T-RFLP analysis during winter/spring, and probably were washed out from watershed soils or sediment. Various phylotypes associated with the same phylogenetic division, based on tentative identification of T-RFLP fragments, exhibited diverse seasonal patterns, suggesting that ecological roles of Delta bacterioplankton were partitioned at the genus or species level.