Eukaryotic ultraphytoplankton (<5 mu m) are an important component of phytoplankton populations, Dot blot hybridisation analysis using class level 16S rRNA gene probes as well as clone libraries were used to investigate the diversity of these ultraphytoplankton during a 15 mo period (2003 to 2004) in the Gulf of Naples. Hybridisation data showed the presence of 3 main classes, Cryptophyceae, Chrysophyceae and Prymnesiophyceae, along with lower signals from the Pelagophyceae. Clone libraries also contained these 4 classes as well as sequences from the Dictyochophyceae, Bacillariophyceae and Prasinophyceae. However, the Prymnesiophyceae gave the dominant hybridisation signal and constituted the majority of each clone library. Their diversity, with a total of 190 sequences belonging to 114 operational taxonomic units (OTUs), probably allows them to dominate the ultraphytoplankton throughout the whole year under differing environmental conditions. Over 100 of these OTUs were unique to different libraries, suggesting a succession of different taxa during the year. The Cryptophyceae were present most of the year with 1 OTU, corresponding to a Plagioselmis prolonga strain from the Gulf of Naples, being the dominant taxon (28 % of sequences). A striking result was the high hybridisation signal from the Chrysophyceae, which showed a preference for the summer months. The Pelagophyceae were present between December and March. Most (80 %) of the sequences found in the clone libraries were not identical to available 16S rRNA gene sequences, indicating a high amount of hidden diversity for these algal classes. However, sequences from Prasinophyceae Clade II (Mamiellales) were not detected in the clone libraries.