Alexandrium ostenfeldii is a thecate, mixotrophic dinoflagellate recently linked to a novel suite of toxins called spirolides. This study provides the first description of the regional distribution of A. ostenfeldii in the Gulf of Maine (GOM), and the first report and analysis of spirolide toxicity in A. ostenfeldii in waters south of Nova Scotia. Morphological examination of cells in field samples and of clonal cultures isolated from several stations in the GOM confirmed the presence of A. ostenfeldii. A genus-specific antibody probe, and an A. ostenfeldii species-specific oligonucleotide probe labeled these cells; a probe specific for the North American A. fundyense/tamarense/catenella species complex did not label A. ostenfeldii cells. Cell size ranged from 20 to nearly 90 Âµm, and most cells contained food vacuoles, with a total vacuole size from 1 to 48 Âµm. The hydrographic forcings controlling the distribution of A. ostenfeldii in the GOM are quite similar to those acting on the A. fundyense population at the same time of the year. The highest concentrations of A. ostenfeldii were observed nearshore, to the east of Penobscot Bay, at times with an offshore-turning branch of high cell concentration to the south of Penobscot Bay. Casco Bay appears to be an area of accumulation for A. ostenfeldii cells advected toward shore from the core of the population to the northeast. Concentrations of A. ostenfeldii were generally higher at the surface than deeper, except at locations where the pooling of lower-salinity water at the surface may have led to the subduction of the population flowing in from the east. PSP toxins were detected in field populations containing A. ostenfeldii and A. fundyense, but not in A. ostenfeldii cultures isolated from the GOM. Spirolide toxins were found in 36 of 60 field samples. More than 83% of samples containing A. ostenfeldii cells had one or more of spirolide congeners A, B, C2 and D2. The total concentration of spirolides per cell at each station where A. ostenfeldii was detected ranged from non-detectable to 282 fmol cell-1. A. ostenfeldii cultures originating from different locations showed a great diversity in spirolide content and composition. All cultures contained spirolides, ranging from 28 to 113 fmol cell-1. Spirolide congeners desMeC, C and D were present in some cultures, but were not detected in any of the field samples. Based on differences and similarities between their toxin profiles, five toxin phenotypes were identified. The highest per cell spirolide contents in the cultures were nearly two times lower than the highest levels observed in field samples. Currently there is no routine monitoring for spirolide toxins in shellfish in the region, but it may be necessary eventually to expand ongoing toxin monitoring in the GOM to include analysis for spirolides.