Serine and glycine biosynthesis in yeast proceed by two pathways; a "glycolytic" pathway, using 3-phosphoglycerate, and a "gluconeogenic" pathway, using glyoxylate. We used a mutation in the cat1 gene to abolish the glucose-repressible "gluconeogenic" pathway and re-isolated two mutants, serl and ser2, in the "glycolytic" pathway. The set1 mutation corresponded to phosphoserine transaminase and ser2 to that of phosphoserine phosphatase. Mutagenesis of a serl set2 cat1 triple mutant facilitated the isolation of a mutation in a new gene, SERIO. SERIO appears to be part of a pathway which, under normal growth conditions, is less important in serine biosynthesis. The set1 set2 serlO triple mutants were totally serine auxotrophic on glucose media but serine prototrophic during growth on non-fermentable carbon sources. This phenotype was used to select for possible regulatory mutants that synthesize serine by the gluconeogenic pathway even in the presence of glucose, e.g., with a non-glucose repressible glyoxylate cycle. In an alternative approach to isolate such mutants URA3 and TRP1 expression were placed under the control of the glucose-repressible FBP1 (fructose-l,6-bisphosphatase) promoter. Although both systems resulted in strong selection pressure we could not isolate constitutively derepressed mutants. These results indicate that transcription of glucose-repressible gluconeogenic enzymes is mainly dependent on positive regulatory elements.