Two new cyclic depsipeptides, pipecolidepsins A and B (1 and 2), have been isolated from the sponge Homophymia lamellosa collected off the coast of Madagascar. Their structures were determined by a combination of NMR experiments and by LC-MS analysis of the amino acid fragments obtained by hydrolysis and derivatization using Marfey's reagent. In addition to several common amino acids, these peptides contain unusual residues, including 2-amino-3-hydroxy-4,5-dimethylhexanoic acid, 3-ethoxyasparagine, 3,4-dimethylglutamine, 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, and 3-hydroxyaspartic acid as well as a terminal 3-hydroxy-2,4,6-trimethylheptanoic acid residue. Pipecolidepsins A and B displayed cytotoxic activity against a panel of different human tumor cell lines.