Marine picoeukaryotes, referred as organisms smaller than 3 micrometers in size, are widespread in marine environments and contribute significantly to global primary productivity. Despite their ecological importance, picoeukaryotes have come to the attention only in the last few years, with the development of new cultivating approaches and molecular techniques. However, culturing misses a lot of species due to their inability to grow in artificial media. Molecular techniques based on 18S-rRNA or 16S-rRNA gene sequence analysis reveal taxonomic diversity, distribution and abundance of marine microbiota, but provide no data about physiological features of certain phylogenetic groups and their particular contribution to the net activity of the community. The proposed study is aimed at discovering the major contributors to the microbial food web via their highly expressed genes. There are two primary components of this project: (i) construction of normalized cDNA libraries from natural picoeukaryotic assemblages and creating expressed sequence tag (EST) databases, and (ii) construction of a large-insert environmental DNA (metagenomic) library from the same environment as (i). EST data sets will allow an understanding of how members of the natural picoeukaryotic co mmunity experience their environment. Moreover, EST data will be used to design probes based on the genes managing most ecologically important metabolic pathways, such as marine carbon cycling. Screening the metagenomic library using these probes will iden tify the genomic fragments from which these genes are expressed. Screening the same library by 18S rRNA gene probes and full sequencing of chosen fragments will allow phylogenetic identification of picoeukaryotes engaged in key biogeochemical processes and will provide novel sequence information from these ecologically important species.