Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated. Molecular phylogenetic surveys of rRNA genes have altered the perspective on naturally occurring microbial diversity and distribution (for a review, see references 11, 19, and 32). Despite a greater understanding of microbial identity, distribution, and abundance, rRNA-based gene surveys have provided little information about the biological properties of many planktonic microbes, especially groups for which there are no cultivated representatives. In addition, although rRNA microheterogeneity (variation within highly related rRNA sequence clusters) has been observed in virtually all taxa and environments sampled via rRNA gene surveys, the significance of this phenomenon remains uncertain. Recent advances in genomic sequencing techniques and the development of methods for cloning large genome fragments into fosmid (35,41,42,45) or bacterial artificial chromosome (5, 6, 38) vectors now provide the means to characterize the gene content (41), metabolic potential (5), and population genetics (41) of uncultivated microorganisms, otherwise known solely by an rRNA sequence. The utility of such genomic approaches and their applicability to diverse questions in microbial ecology have only begun to be explored and exploited.Members of the Archaea (48) are much more diverse and widespread than previously suspected. Representatives have now been detected in terrestr...