The absolute configuration of the 2-amino-4-methyl-hex-5-enoic acid isolated from Boletus was shown to be 2S, 4S, by an unambiguous synthesis of its dihydro derivative from 2(S)(-)-2-methylbutan-1-ol. We have previously reported the isolation of 2-amino-4 methyl-hex-5-enoic acid (1) [1, 2] and shown that the asymmetric center at carbon-2 was S but could not establish the configuration at carbon-4 by physicochemical methods. We have now synthesized (2S,4S)-2-amino-4-methyl hexanoic acid (2) unambiguously from 2(S) methylbutan-1-ol and acetylamino malonate [3,4], followed by resolution of the carbon-2 center of the acetylaminomethylhexanoic acid by hog kidney acylase I [5, 6]. The synthetic product was identical in all respects with the dihydro compound (2) obtained from the natural product (1) by catalytic hydrogenation. As catalytic hydrogenation of the Δ5 double bond at room temperature will not change the chirality at C-4, the absolute configuration of 1 is established as (2S,4S)-2-amino-4-methylhex-5-enoic acid. The unambiguous synthesis of sterically pure 2 also establishes the stereochemistry of the naturally occurring amino acid, homoisoleucine, which had been isolated by Fowden and Smith [7] from Aesculus californica. Fowden et al. [8] had already proposed the 2S,4S configuration for homoisoleucine on the basis of a comparison of its solubility, ORD and CD with those of isoleucine.