Despite the fact that bacterioplankton play a key role in oceanic biogeochemical cycles, their biogeographical distributions remain largely unknown.  We examined prokaryotic biogeography in the North Atlantic gyre between Barbados and Cape Verde using genetic and genomic techniques. The longitudinal gradient is characterized by decreasing surface water productivity and consequent deepening of the deep chlorophyll maximum layer from west to east. Bacterial, archaeal, and diazotroph assemblages were investigated through combinations of clone library analysis, gene-specific and metagenomic microarrays, and quantitative PCR to describe trends in the abundance and diversity of the prokaryotic assemblage at the community and functional group level. Bacteria and archaea demonstrated remarkable transect-wide heterogeneity at the 16S rRNA level in near-surface waters; in contrast, the diazotroph assemblages were made up of some phylotypes endemic to different habitats and others widely distributed. Diversity of diazotrophs by DNA-based analyses was much higher than detectable as gene transcripts. Small metagenomic and transcriptomic libraries prepared from the West and East Atlantic contained different sequences, suggesting that subtle environmental pressures can have important effects on genetic potential and active processes within the basin. Moreover, these results demonstrate that factors selecting for dominant components of microbial assemblages exist at the basin scale; however, those selecting for organisms with specific functions may occur at smaller spatial scales. Finally, our results demonstrate that selection for functional capacities (e.g. nitrogenases) may occur on spatio-temporal scales that are consistent with dispersal of taxa in open ocean bacterioplankton.