The absolute stereochemistry of amphidinolide B (1), a potent cytotoxic 26-membered macrolide isolated from the cultured marine dinoflagellate Amphidinium sp., has been established as 8S, 9S, 11R, 16R, 18S, 21R, 22S, 23R, and 25S, on the basis of enantiospecific synthesis of a degradation product (2).