Genetic and biochemical analyses were conducted on fatty acid mutants of yeast deficient for Delta(9)-desaturase activity in the production of palmitoleate and oleate. Two genetic loci were observed and two others are inferred; three of these were represented by respiratory-deficient (petite) strains. All strains were incapable of converting palmitate to palmitoleate and stearate to oleate whether the direct precursor or acetate was followed. All strains were capable of acylating both de novoproduced fatty acids and oleate taken up from the medium into phospholipids and neutral lipids. Two revertants were analyzed which differed in their ability to produce palmitoleate and oleate.