The content of eicosatetraenoic (C20:4) acid in Plexaura homomalla lipids was determined by transesterification with sodium methoxide and gas-liquid chromatography of the resulting fatty acid methyl esters. The C20:4 methyl ester constituted 22.6 % of the non-polar fatty acid esters of variant R lipids, 33.9 % of the non-polar fatty acid esters of variant S lipids, and 0.4% of the dry weight in both variants. The position and stereochemistry of the double bonds in the C20:4 acid was determined by partial reduction to a C20:1 acid mixture, separation of cis and trans C20:1 methyl esters on AgNO3 impregnated silicic acid thin layer plates, and oxidative cleavage of the C20:1 methyl ester mixture with permanganate-periodate. The double bonds were shown to be at Positions 5, 8, 11, and 14 with less than 2.4 % of the molecules having a trans double bond. Therefore, more than 97% of the C20:4 acid in the lipids of both variant S and variant R of P. homomalla is arachidonic acid, and the difference in stereo-chemistry at Position 15 of the P. homomalla prostaglandins ((15R) for variant R, (15S) for variant S) cannot be reasonably explained by a stereochemical difference of a C20:4 precursor fatty acid.