The role of the Ca2+-calmodulin system in the control of cellular proliferation and tumor formation has been of great interest, with the well-known calmodulin antagonist W-7 found to inhibit proliferation of Chinese hamster cells and formation of mouse skin tumors. In continuing studies on bioactive substances from tunicates, pseudodistomins A (1) and B (2), potent antineoplastic piperidine alkaloids with calmodulin antagonistic activity, were isolated from the Okinawan tunicate Pseudodistoma kanoko, representing the first isolation of piperidine alkaloids from marine sources. The tunicate (400 g wet weight) was extracted with methanol-toluene (3:1), and the chloroform extract of the aqueous layer (showing cytotoxicity against L1210 cells) was purified via flash silica gel chromatography and reversed-phase HPLC to yield a ~1:1 mixture of 1 and 2. Due to their air-oxidation susceptibility and double bond isomerization, further purification used their acetates (3 and 4) obtained by acetylation and HPLC, with yields of 0.012% and 0.018% (wet weight) for 3 and 4, respectively. Structure determination (via 1H/13C NMR, COSY, 2D 1H-13C shift correlation, catalytic hydrogenation, NOE, and CD spectroscopy) revealed pseudodistomin A (1) is 2(R)-(trideca-3'(E),5'(Z)-dienyl)-4(R)-hydroxy-5(S)-aminopiperidine and pseudodistomin B (2) is 2(R)-(trideca-3'(E),5'(E)-dienyl)-4(R)-hydroxy-5(S)-aminopiperidine, with absolute configurations 2R, 4R, and 5S. Biological assays showed 1 and 2 exhibit cytotoxicity against L1210 (IC50: 2.5, 0.4 μg/mL) and L5178Y (IC50: 2.4, 0.7 μg/mL) murine leukemia cells, and inhibit calmodulin-activated brain phosphodiesterase (IC50: 3×10-6 M), being ~3 times more potent than W-7. In vivo antitumor studies are ongoing.