In the course of our search for biologically active substances from marine organisms, we isolated a potent cytotoxic polyketide named callystatin A (1) from the marine sponge Callyspongia truncata collected at Goto Islands in Nagasaki Prefecture. An acetone extract of the titled fresh sponge (1.0kg) showing cytotoxic activity was subjected to bioassay-guided separation to furnish 1 (1.0mg, 1.0×10^<-4>% from the fresh sponge) as an active substance. Callystatin A (1) exhibited extremely potent cytotoxicity against KB cells at IC_<50> 10pg/ml. The plane structure of 1 having α,β-unsaturated δ-lactone, two conjugated diene, and β-hydroxyketone portions was elucidated by detailed NMR analysis. The absolute configuration of C-19 linked to a hydroxyl group was determined to be R by modified Mosher method. The stereochemistry around β-hydroxyketone portion was established as 16R, 18S, and 20S by comparison of the NMR data of 1 with those of (-)-ebelactone, 12-epi-(-)-ebelactone, and some related polyketides. Additionally, the unidentified 5R and 10R configurations were revealed by comparative CD analysis of 1 and the four model compounds (2, 3, 16, 17). The model compounds were synthesized through high E-selective Wittig olefination between an aldehyde 9, whose acetal moiety was easily converted to α,β-unsaturated δ-lactone, and allylic tributylphosphorus ylides 14, 20 as a key step. In connection with callystatin A (1), several related antitumor antibiotics were isolated from actinomyces and their plane structures are clarified. To our knowledge, this is the first example of elucidation of the absolute stereostructure among the related cytotoxic polyketides.