Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It is produced by Streptomyces sp. MRI142 and has a novel structure of a tetramic acid derivative with a long alkyl side chain. The side chain is polyhydroxylated and acyclic except for a tetrahydropyran ring moiety. Determination of the absolute configuration of 1 is very important for further studies, but there has been a little imformation about the stereochemistry of 1. Untill now, we have determined the absolute configuration of chromophore moiety (fragment A) and the relative stereochemistry around the tetrahydropyran ring. In this experiment, absolute configurations of remaining 26 chiral centers in 1 were chemically elucidated. First, two small fragment molecules were prepared from 1 or its methyl ether (5) by degradation experiments mainly using NaIO_4, and thier absolute structures were assigned as (R)-3-hydroxydodecanoic acid (2) and (R)-1,2,4-butanetriol tribenzoate (3). Next, an acyclic fragment molecule (fragment B) with 13 chiral centers was obtained from 1 by NaIO_4 oxidation, and its relative stereochemistry was elucidated by J-based configuration analysis. By analyzing coupling constants of ^3J_<H,H> and ^<2,3>J_<C,H> obtained from E. COSY, HETELOC and phase-sensitive HMBC spectra and partly supplemented ROE data, the relative configuration of fragment B was established. Finally, by futher J-based configuration analysis using a fragment molecule prepared from 5 with 28 chiral carbons, all relative configurations in the alkyl side chain of 1 were clarified. By connecting these relative configurations with the absolute configurations of the first four fragment molecules, absolute stereochemistry of 1 was fully determined. Aflastatin B (6), a minor component of 1, was purified and its structure was identified as a N-demethylated derivative of 1. Blasticidin A (7) was found in 1955 in the culture broth of S. griseochromogenes, and its isolation and detailed physicochemical properties were reported in 1968. However, the structure of 7 was unknown. Since close homology was noticed between the physicochemical properties of 1 and 7, the biological activity of 7 was examined, and 7 was found to inhibit aflatoxin production. This finding prompted us to investigate the structure of 7, and we determined the structure of 7 as a structurally related compound of 1. The biological activities of aflastatin B (6) and blasticidin A (7) were assessed, and it was shown that these compounds inhibited the aflatoxin production as strongly as 1.