A suite of biologically active compounds, fast-acting toxins called spirolides, were recently isolated and characterized from shellfish and plankton samples collected from southeastern Nova Scotia, Canada. Circumstantial evidence from natural plankton assemblages has linked spirolides to gonyaulacoid dinoflagellates, particularly athecate cells (GB-42) that are derived from motile stages from this group. After repeated attempts to isolate spirolide-producing taxa from enriched size-fractionated field samples, numerous isolates of potential source organisms, including Alexandrium ostenfeldii (Paulsen) Balech et Tangen, Alexandrium tamarense (Lebour) Balech, Fragilidium subglobosum (von Stosch) Loeblich III, Gonyaulax spinifera (Claparède et Lachmann) Diesing, Protoceratium reticulatum (Claparède et Lachmann) Bütschli, and Scrippsiella trochoidea (Stein) Loeblich III were successfully cultured. Among all cultured dinoflagellate species harvested in late exponential growth phase and analyzed for spirolide content, only A. ostenfeldii was found to produce these compounds. Detailed morphological examination of field specimens and cultured cells of A. ostenfeldii by Nomarski and epifluorescence optical microscopy and by scanning electron microscopy showed that in most major aspects specimens from the western Atlantic coast closely resemble this species as described from Scandinavian waters. Key diagnostic features are the globose morphology and the presence of a large kidney-shaped ventral pore at the margin of the first apical (I') plate. Analysis by liquid chromatography with detection by tandem mass spectrometry (LC–MS/MS) of the spirolides in an isolate from Ship Harbour, Nova Scotia, indicated that only the C/D type was present in significant amounts. The principal spirolide (90% molar), a desmethyl-C derivative (MW 691), was present at a mean concentration of 54 pg cell−1. This profile is consistent with that found in bulk plankton samples collected from the same site, thereby confirming A. ostenfeldii as the primary source of spirolides in this region.