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The principal sex pheromone component produced by females of the cabbage moth, Mamestra brassicae , is derived from the monounsaturated fatty acid, Z 11-16:1, whereas two additional trace components are derived from E 11-16:1 and Z 9-16:1. This report presents the isolation and analysis of cDNAs encoding pheromone gland-specific acyl-CoA desaturases implicated in the production of these unsaturated fatty acids (UFAs). Comparisons of the encoded amino acid sequences of four cDNA fragments isolated by degenerate PCR from cabbage moth pheromone glands established their orthology with previously characterized noctuid desaturases as follows: Mbra LPAQ, belonging to the pheromone gland-specific LPAQ desaturase lineage having Δ11 regioselectivity, Mbra KPSE-a and Mbra KPSE-b, belonging to the pheromone gland-specific KPSE desaturase lineage having Δ9 regioselectivity and a substrate preference for palmitic acid (16:0) over oleic acid (18:0), and Mbra NPVE, belonging to the NPVE desaturase lineage having Δ9 regioselectivity and a substrate preference 18:0>16:0. Full-length cDNAs corresponding to the two most abundantly expressed pheromone gland-specific desaturase transcripts, Mbra LPAQ and Mbra KPSE-b, were isolated and assayed for their ability to genetically complement the UFA auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae . The Mbra LPAQ desaturase restored UFA prototrophy and GC–MS analysis identified Z 11-16:1 and Z 11-18:1 as the predominant UFAs produced. Surprisingly, Mbra KPSE-b failed to complement the ole1 mutation, although it shares >98% amino acid sequence similarity with other noctuid KPSE desaturases that do. Site-directed mutagenesis of either or both of two nonconservative amino acid substitutions restored functionality to the Mbra KPSE-b protein, although GC–MS analysis revealed that neither reversion resulted in the characteristic KPSE substrate preference for 16:0.