In late 2004, the BIOSOPE cruise sailed between the equatorial influenced waters off the Marquesas Islands and the nutrient enriched waters of the Chilean upwelling. Along the way, it explored the Southeast Pacific gyre centred around Easter Island, which is probably the most oligotrophic oceanic region on earth. During this cruise, we undertook a vigorous effort to isolate novel photosynthetic picoplanktonic eukaryotes. Two strategies were attempted on board: enrichment of filtered samples with culture medium and sorting of specific populations by flow cytometry based on size and chlorophyll fluorescence. Over 1900 pre-cultures were started and then further purified by flow cytometry, serial dilution or pipette isolation to yield a total of 212 strains. These strains were characterized morphologically and for more than 50% of them, genetically, through partial sequencing of the 18 S rRNA gene. Among the characterized strains, the largest number belongs to stramenopiles (Heterokontophyta) with a record of 38 strains belonging to the species Pelagomonas calceolata (Pelagophyceae). Strains from the recently described genera Bolidomonas and Florenciella have been re-isolated for the first time since their description. Two other abundant groups are the Chlorophyta, especially Prasinophyceae, and the Haptophyta, especially the genera Phaeocystis and Emiliania. A limited number of heterotrophic flagellates have also been isolated, all of them belonging to groups containing known species. Finally, over a dozen of unicellular cyanobacterial Synechococcus strains have been obtained, some forming unusual short chains. Overall our strategy was quite successful since it allowed us to isolate a large number of picoplankton strains. Still it failed in two respects. First, apparently very few novel taxa have been obtained. One set of strains is related to Prasinoderma coloniale (Prasinococcales, Prasinophyceae) but their sequences are sufficiently different from the latter to probably belong to a new genus or species. The sequences of two other strains, unfortunately later lost, were phylogenetically affiliated to stramenopile environmental sequences, probably corresponding to a new algal class. Second, very few strains have been obtained from the very oligotrophic central gyre itself. In order to be successful, future work in similar waters should probably combine flow cytometry sorting with culture media and cultivation approaches specifically developed for oligotrophic water species.