Lipophilic phycotoxins in size-fractionated plankton net tows (20 μm mesh-size) were measured on-board during a month-long oceanographic cruise in North Sea coastal waters. Tandem mass spectrometry coupled to liquid chromatography (LC–MS/MS) was employed for the detection and quantification of a broad spectrum of known and putative phycotoxins. For pectenotoxins (PTXs) the following ion masses ([M + NH4]+) were monitored: m/z 876 for PTX-2, m/z 892 for PTX-11 and PTX-13, and m/z 874 for PTX-12 and PTX-14. The PTX levels in net plankton were highest along the Danish north coast, but levels over 50 ng per net tow were also detected on the southern Scottish East coast and in the northern Skagerrak. Abundance of PTXs was highly correlated with the occurrence of the marine dinoflagellate Dinophysis spp. Whereas in the eastern North Sea PTX-2 was the most abundant PTX, in the western North Sea PTX-1 was the major component, but it was also present in lower proportions in the Norwegian and Danish waters than in the western North Sea. Isobaric PTX-11 was absent or only detected at trace levels throughout the entire cruise, and PTX-13 and PTX-14 were not detected at all. The identity of PTX-1 was confirmed by comparison of retention time and mass spectrum of the North Sea phytoplankton sample to PTX-1 previously isolated from shellfish. Statistical analysis showed the best correlation between the occurrence of PTX-1 and Dinophysis acuminata cell concentration. Nevertheless, we could not rule out the possibility of metabolic transformations of PTXs by organisms that have grazed upon Dinophysis. Such biotransformations could conceivably occur in heterotrophic dinoflagellates or ciliates, or even via oxidation in copepod fecal pellets. In any case, this study confirmed the presence of PTX-1 in the plankton and is the first definitive report of this toxin in the North Sea.