A novel tridecadepsipeptide, theonellamine B, has been isolated from an Okinawan marine sponge of the genus Theonella as a specific inhibitor against Na,K-ATPase. Its structure has been determined to be a lactone of methoxyacetyl-&Val-D- ~Leu-~-Thr-8-Ala-~-Leu-~-MeIle-~-Ala-D-allo-I1e-~-MeVal-I,-MeAla-8-Ala-~-Leu-L-allo-MeIle on the basis of chemical reaction and spectral data including two dimensional homo- and heteronuclear shift correlation experiments.Though large numbers of novel natural products have been isolated from marine sponges, 3 only four bioactive peptides have been reported.4 In our continuing search for physiologically active substances from marine sources, we recently found several enzyme modulators from Okinawan sponges. 5 This report deals with the isolation and structure elucidation of theonellamine B, an unusual tridecadepsipeptide with a 37 membered ring. This is the first peptide that inhibits Na,K-ATPase (58/a inhibitory concentration in pig brain Na,K-ATPase, 7~10~~ M).