Spirolides are pharmacologically active macrocyclic imines discovered in shellfish and plankton size-fractions collected from near-surface waters along the southeastern coast of Nova Scotia (NS), Canada. These compounds are most prevalent in the water column within plankton size-fractions from 21 to 56 μm during late spring. Analysis of bulk plankton material by liquid chromatography combined with ion-spray mass spectrometry indicated that gonyaulacoid dinoflagellates were domin-ant members of the plankton assemblage in these fractions when spirolides were abundant at Graves Shoal, NS, but that spirolides disappeared from the water column after a shift to diatom dominance. Spirolide composition in the plankton was rather consistent over time and among different sizefractions within a site, but the profile was radically different between sites in Nova Scotia. At Ship Harbour, NS, spirolides A, B, C and desmethyl-C were the major constituents, whereas at Graves Shoal, the primary spirolide derivatives were B, D and an isomer D2. Correlation analysis of spirolide abundance and the occurrence of particular taxa in size-fractionated plankton showed that the highest correlations were with Alexandrium species (r2 = 0.93) and GB42 cells (r2 = 0.83). These latter cells are now recognized as athecate forms of gonyaulacoid dinoflagellates, primarily of Alexandrium ostenfeldii. By analysis of size-fractionated plankton and comparison with cultured plankton isolates from spirolide-rich areas, we have substantiated the primary role of A. ostenfeldii as the source of spirolides in the water column.