A fourth cytotoxic macrolide, amphidinolide D[1], together with known amphidinolide B[2], has been isolated from a different batch of the cultured dinoflagellate Amphidinium sp., which was symbiotically associated with an Okinawan flatworm Amphiscolops sp. Two-dimensional nmr experiments including 1H-detected heteronuclear multiple-bond correlation (HMBC) resulted in the structure assignment of 1 and structure revision of the diene moiety (C-13-C-15) of amphidinolide B.