ABSTRACT Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2 ^Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b ₅ protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b ₅ domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b ₅ s were coexpressed with the desaturase. Coexpression of a cytochrome b ₅ from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b ₅ proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.