Planktonic picoeukaryotes are important players in coastal marine food webs but little is known about their diversity. Here we report the picoeukaryotic diversity in Blanes Bay (NW Mediterranean) by retrieving environmental 18S rDNA sequences and by obtaining stable cultures. Four genetic libraries (one per season) were constructed and 383 clones analyzed, yielding 176 distinct sequences. The diversity of picoeukaryotes was very large, both at higher and lower phylogenetic levels. Novel alveolates-I (36% of clones), dinoflagellates (17%), novel stramenopiles (10%), prasinophytes (5%) novel alveolates-II (5%), and cryptophytes (4%) were the better represented phylogenetic groups. Nineteen additional groups were found at <3% clonal abundance. The four genetic libraries were dominated by the above-mentioned groups, implying a relative stability at high taxonomic level, but identical sequences were seldom found in consecutive dates, suggesting fast temporal changes of picoeukaryotic populations. Coastal and open sea picoeukaryotes were similar, but the representation of groups varied between habitats. The culturing effort revealed that some groups were well represented in clone libraries and in cultures (prasinophytes), others were found by both approaches but often with different sequences (cryptophytes), and others were found only in cultures (bicosoecids) or in clone libraries (novel alveolates and stramenopiles). Our data confirm that molecular approaches, such as cloning and sequencing 18S rRNA genes, are a necessary first step to study picoeukaryotic diversity. These results will aid to focus future research, most likely based on new and imaginative culturing efforts and the design and application of specific molecular probes.