A study has been made of the acid pyrophosphatase, carbamoylphosphate ornithinecarbamoyl(CPOC) transferase, invertase, and malic dehydrogenase activities of Saccharomyces cerevisiae grown in a defined medium containing either an optimal or a suboptimal concentration of d-biotin; the effect of supplementing the biotin-deficient medium with l-aspartic acid and/or oleic acid was also examined. Acid pyrophosphatase, invertase, and malic dehydrogenase activities were frequently higher in yeast grown under conditions of varying biotin deficiency than in biotin-optimal yeast. It is concluded that synthesis of these enzymes is not specifically biotin dependent and that the different activities in biotin-deficient yeast are probably the result of an alteration in the normal induction and repression mechanisms and possibly also of a decreased synthesis of repressor substances. CPOC transferase activity was always low in yeast grown in biotin-deficient media as compared with biotin-optimal yeast, and it is suggested that synthesis of this enzyme may be specifically biotin dependent. Yeast grown in biotin-deficient medium, with or without oleic acid, was shown to be more fragile than yeast grown in biotin-optimal medium or in biotin-deficient medium supplemented with aspartic acid; these observations are discussed in relation to changes in the cell wall metabolism of yeast accompanying biotin deficiency. © 1962.