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The absolute configurations at 12 chiral centers in amphidinolide C (1), a potent cytotoxic 25-membered macrolide isolated from a marine dinoflagellate Amphidinium sp,, were determined to be 3S, 4R, 6R, 7R, 8R, 12R 135, 16S, 20R, 23R, 24R, and 29S by combination of NMR analyses, degradation experiments, and synthesis of the C-1-C-7 segment.