Hapalosin (2), a novel cyclic depsipeptide from the blue-green alga (cyanobacterium) Hapalosiphon welwitschii W. & G. S. West (Stigonemataceae), shows MDR-reversing activity. Its structure has been determined to be (3S,4R,8R,9S,12S)-9-benzyl-4-heptyl-8-hydroxy-l2-isopropyl-3,l0-dimethyl-l,5-dioxa-l0-azacyclododecane-2,6,1 l-trione by a combination of spectroscopic and chemical methods. The carbon skeleton of 2 was determined by two-dimensional ¹H-¹H and ¹H-¹³C NMR correlation experiments and confirmed by mass spectral analysis. The relative and absolute stereochemistry was determined by coupling constant data and Mosher analysis of 2 and three degradation products obtained from acid hydrolysis of 2. Agents that are capable of overcoming P-glycoproteinmediated multidrug resistance (MDR) have potential use in the treatment of cancer patients undergoing chemotherapy. Two types of anti-MDR drugs are desirable, viz. (1) cytotoxic drugs which are equally efficacious toward drug-sensitive and drug-resistant tumor cells, and (2) MDR-reversing agents that are able to potentiate the cytotoxicity of common antitumor drugs like vinblastine and adriamycin toward drug-resistant cells. In screening extracts of blue-green algae (cyanobacteria) for anti-MDR activity, about 1% of the extracts exhibited MDR-reversing activity. The lipophilic extracts of two strains of Hapalosiphon welwitschii W. & G. S. West (UH IC-52-3 and UTEX B1830) reversed MDR in a P-glycoprotein-overexpressing, vinblastine-resistant subline (SKV-LB1) of a human ovarian adenocarcinoma line (SKOV3). Some of the MDR-reversing activity was attributed to an unusual indole alkaloid, N-methylwelwitindolinone C isothiocyanate (1), but much of the activity was due to a completely different compound. We report here the isolation and structure determination of a new cyclic depsipeptide, hapalosin (2), which accounts for this remaining MDR-reversing activity.