The structural genes coding for the two kinds of subunits of phosphofructokinase in yeast have been cloned previously (Heinisch 1986). The coding regions were defined by S1-mapping. They were disrupted in vitro by insertion of a LEU2-marker. These constructions were then used for substitution of the respective chromosomal copies. That the disruption of the PFK-genes had in fact occurred was confirmed by Southern blot analysis. Furthermore, in Northern blots shorter transcripts were detected in the respective disruption mutants. Using polyclonal antibodies the alpha-subunits were not detectable in pfk1-disruptions whereas the beta-subunits were undetectable in pfk2-disruptions. Physiological characterization showed that the single disruption mutants still fermented glucose to ethanol and CO2. They accumulated fructose-6-phosphate and glucose-6-phosphate over wild type levels and showed decreased levels of fructose-1,6-bisphosphate. In addition an accumulation of sedoheptulose-7-phosphate was observed, a metabolite not detectable in wild type cells. A haploid yeast strain containing both disrupted copies of the PFK-genes is not capable of growing on rich medium containing 2% glucose. The accumulation of glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate is much more pronounced in such mutants, whereas the fructose-1,6-bisphosphate concentration decreases below the level of detection.