Mutants lacking fructose 1,6-bisphosphatase activity have been isolated from Saccharomyces cerevisiae and genetically purified. Mutants were unable to grow on gluconeogenic carbon sources. Revertants that grew on glycerol have regained the fructose 1,6-bisphosphatase activity, showing that there is no bypass in yeast for this enzymatic step. No significant differences were found in the growth of the mutants and the parental strains in other carbon sources. Other mutants lacking fructose 1,6-bisphosphatase activity but pleiotropically affected in the derepression of several enzymes sensitive to catabolite repression were also isolated. All the mutants isolated were of nuclear origin and defined three complementation groups.