The naturally occurring sophorolipids synthesized by Candida bombicola possess—despite their overall heterogeneity—little variation in the length of the lipid tail. The range is limited to C16–C18 fatty acids and is governed by the specificity of a cytochrome P450 monooxygenase. However, incorporation of fatty acids differing from the conventional C16–C18 range could broaden up the application potential of sophorolipids. The incorporation of medium-chain fatty acids should render the molecules more hydrophilic and consequently improve their water solubility. Two strategies to circumvent this C16–C18 preference are described in this paper. The first one skips the controlling action of the cytochrome P450 enzyme by supplying the yeast with already hydroxylated substrates, while the other method is based on the deception of the enzyme by presenting it substrates structurally resembling stearic acid. This later strategy can be applied to create very specific tailor-made sophorolipids when combined with post-fermentive modification. Biotechnol. Bioeng. 2011; 108:734–741. © 2010 Wiley Periodicals, Inc.