Oligonucleotide probes based on small subunit 18S rRNA variable region sequences, were designed and used to discriminate groups of marine green ultraphytoplankton of similar size and morphology. Hybridization of probes was visualized using a combination of biotinylated oligonucleotides and fluorescein labeled avidin. Increased fluorescent signal was generated by building a complex of biotinylated probe, fluorescein labeled avidin and biotinylated anti-avidin. Cells of one phylogenetic group were easily differentiated from morphologically similar cells of a different phylogenetic group. Design of specific probes allowed identification of Mantoniella squamata, Micromonas pusilla, Pseudoscourfieldia marina and the micromonadophyte clone CCMP1194. This technique should be readily adaptable to open ocean samples and should greatly facilitate the description and quantification of ultraphytoplankton in the open ocean.