The effect of linolenic acid (C18:3ω3) on the development of Strongyloides ratti first-stage larvae (L1) in culture was studied. The fatty acid composition of S. ratti free-living generations was analyzed by gas chromatography. L1 had abundant linoleic acid (C18:2ω6) but its proportion decreased with development. On the contrary, eicosapentaenoic acid (C20:5ω3) and C20:4ω3 were prominent in the filariform larva (L3). Because C20:5ω3 is generally synthesized from C18:3ω3 via C20:4ω3, the high ratio of C20:5ω3/C18:3ω3 of L3 in all the free-living generations suggested that polyunsaturated fatty acid metabolism, particularly the ω-3 series, and eicosanoids produced had important roles in the development of S. ratti L1.