ABSTRACT A cDNA encoding pheromone Δ9 acyl‐CoA desaturase, SlitKPSE was isolated from sex pheromone gland of the tobacco cutworm, Spodoptera liturawhich uses a diene unsaturated fatty acid (UFA) derivative, Z9E11‐14 : 2 as a major pheromone component. The fulllength open reading frame coding region of SlitKPSE was inserted in a yeast shuttle vector, YEpOLEX, and two kinds of yeast (Saccharomyces cerevisiae) mutant strains were transformed with the recombinant vector. In the desaturase‐deficient ole1 strain, SlitKPSE expressed a complementary enzyme producing two kinds of diene UFAs, more 9–16 : 1 and less 9–18 : 1 at a ratio of 1 : 0.74 exhibiting a typical functional characteristics as one of the pheromone Δ9 acyl‐CoA desaturase lineage group, KPSE, but no Δ9 14C monoene was detectable because of too small amount of 14C saturated fatty acid precursor to be reliably used by SlitKPSE in the transformed cells. However, the another transformed yeast strain elo1 which is deficient of elongase 1, an enzyme converting 14C to 16C hydrocarbon substrate, was supplemented with some myristic acid (14 : 0) in the medium, and produced a significant amount of 9–14 : 1 in due to a much enhanced level of the 14C substrate suggesting that SlitKPSE may be responsible for making the Δ9 double bond on the diene pheromone component.