ABSTRACT: BACKGROUND: With genome sequencing becoming more and more affordable, environmental shotgun sequencing of the microorganisms present in an environment generates a challenging amount of sequence data for the scientific community. These sequence data enable the diversity of the microbial world and the metabolic pathways within an environment to be investigated, a previously unthinkable achievement when using traditional approaches. DNA sequence data assembled from extracts of 0.8 mum filtered Sargasso seawater unveiled an unprecedented glimpse of marine prokaryotic diversity and gene content. Serendipitously, many sequences representing picoeukaryotes (cell size <2 mum) were also present within this dataset. We investigated the picoeukaryotic diversity of this database by searching sequences containing homologs of eight nuclear anchor genes that are well conserved throughout the eukaryotic lineage, as well as one chloroplastic and one mitochondrial gene. RESULTS: We found up to 41 distinct eukaryotic scaffolds, with a broad phylogenetic spread on the eukaryotic tree of life. The average eukaryotic scaffold size is 2,909 bp, with one gap every 1,253 bp. Strikingly, the AT frequency of the eukaryotic sequences (51.4%) is significantly lower than the average AT frequency of the metagenome (61.4%). This represents 4% to 18% of the estimated prokaryotic diversity, depending on the average prokaryotic versus eukaryotic genome size ratio. CONCLUSION: Despite similar cell size, eukaryotic sequences of the Sargasso Sea metagenome have higher GC content, suggesting that different environmental pressures affect the evolution of their base composition.