Onchidin B (4) is a cyclic depsipeptide isolated from the pulmonate mollusc Onchidium sp. Its structure was determined by extensive 2D-NMR, FABMS, tandem FAB MS/MS, selective hydrolysis, and synthesis. It contains four alpha-amino acids [two units of N-methyl valine (MeVal), two units of proline (Pro)], four alpha-hydroxy acids [two 2-hydroxyisovaleric acids (Hiv), two 2-hydroxy-3-methylpentanoic acid moieties (Hmp)] and two units of the new beta-hydroxy acid : 3-hydroxy-2-methyloct-7-ynoic acid (Hymo)]. Selective hydrolysis and direct comparison by chiral CC-MS with authentic samples of the alpha-amino and alpha-hydroxy acids allowed us the assignment of the entire absolute stereochemistry of onchidin B. In this way, the alpha-hydroxy acids were found to be (S)-Hiv and (S,S)-Hmp, and the a-amino acids (R)-proline, (S)-proline, and (R)-MeVal. In order to establish the absolute configuration of the new beta-hydroxy acid, Hymo, its four possible stereoisomers were stereoselectively synthesized using chiral N-propionyl oxazolidinones and hex-5-ynal as starting material. Comparison by HPLC-MS of the synthetic samples with the natural Hymo (all derivatized as esters of (-)-(R)-alpha-methoxy-alpha-(9-anthryl) acetic acid), affirmed its absolute stereochemistry as (2R,3R). Thus, onchidin B (4) is cycle [(R)-MeVal-(R,R) -Hymo-(S)-Pro-(S,S)-Hmp-(S)-Hiv-(R)-MeVal-(R,R)-Hymo-(R)-Pro-(S,S)-Hmp-(S)-H . It is formed by a head-to-tail linkage of two halves, each one built by five units and identical sequence. The lack of symmetry of onchidin B is thus due to the presence of one (S)-Pro unit in one half and one (R)-Pro unit in the other. The structural similarity between onchidin B (4) and onchidin (2), both isolated from the same organism, and between the beta-hydroxy acid Hymo (5) and the beta-amino acid Amo (3) found in onchidin is noticed.