Four species of Drosophila (melanogaster, simulans, mauritiana and virilis) were tested for their ability to survive on erg strains of Saccharomyces cerevisiae each having a mutant block at a different reaction in the synthesis of ergosterol. Species capable of completing development on a given yeast mutant strain were tested for egg production and viability, and also for their ability to survive on the mutant yeast for five complete generations. Sterol analyses using gas–liquid chromatography show that the sterol composition of flies closely resembles that of the yeasts on which they are grown, confirming that the steric criteria for sterols capable of a structural role in lipoprotein membranes are relatively broad. Even so, different sterols are not equally efficient in this respect and there are differences between species in their tolerance to the sterols available from each of the yeast mutants. The range of sterols capable of satisfying the micronutrient, as distinct from the macronutrient, requirement is narrower. Growth on two of the yeast mutants (erg-2 and erg-6) leads to developmental arrest in the larval stage due, it is suggested, to a block in the synthesis of ecdysone. The four Drosophila species lack an effective level of Δ8−Δ7 isomerase which is necessary for utilization of zymosterol and other Δ8 sterol precursors of ergosterol. Ergosta-5,7-diene-3β-ol appears to be capable of substituting for cholesterol in the metabolism of Drosophila.