Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase was studied using the protease-deficient and vacuole-defective yeast strain pep4-3. The catabolite inactivation of fructose 1,6-bisphosphatase in pep4-3 was found to have a normal first inactivation step but with a defective second proteolytic step. In contrast, catabolite inactivation of cytoplasmic malate dehydrogenase was normal in pep4-3. These results suggest that the proteolytic pathways utilized in the hydrolysis of the two enzymes may be different and that proteolysis of fructose 1,6-bisphosphatase may require functional vacuoles while proteolysis of cytoplasmic malate dehydrogenase may not.