The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10 7 gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml ؊1 and the maximum number of 912h4 gene copies detected was approximately 340 ml ؊1 . Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.The abundance and biogeochemical importance of marine bacteria has been realized for decades (19). Facilitated by advances in molecular biology techniques, in recent years the taxonomic and phylogenetic identities of marine bacteria have been investigated. Using rRNA genes as phylogenetic markers (18), Giovannoni and coworkers identified previously unknown bacterial phylogenetic lineages in the Sargasso Sea (12), indicating that natural communities of bacteria were not well represented in culture collections. Since that time, studies of the diversity of marine microbial communities have revealed that the majority of bacteria in the ocean are uncultivated and many belong to unexpected taxa (8,11,14,20).With similar molecular methods but aimed at genes encoding enzymes involved in key biogeochemical transformations (sometimes termed functional genes), studies have investigated the diversity and roles of microorganisms involved in specific processes, such as sulfate reduction (9) and nitrogen cycling (31). Biological nitrogen fixation, the reduction of atmospheric dinitrogen to ammonium, is an important process in maintaining the availability of fixed inorganic nitrogen in the biosphere, balancing the opposing process of denitrification. A recent modeling study illustrated the importance of nitrogen fixers to oceanic primary production and argued that nitrogen fixation is the main reason nitrogen availability limits primary production only on short time scales, while phosphorus limits oceanic production over longer time scales (26).Nitrogenase is the multisubunit enzyme that c...