Fructose-1,6-bisphosphatase (FruP2ase) from Saccharomyces cerevisiae is rapidly inactivated upon addition of glucose to a culture growing on non-sugar carbon sources. Under the same conditions the FruP2ases from Schizosaccharomyces pombe or Escherichia coli expressed in S. cerevisiae were not affected. A chimaeric protein containing the first 178 amino acids from the N-terminal half of S. cerevisiae FruP2ase fused to E. coli beta-galactosidase was susceptible to catabolite inactivation. Elimination of a putative destruction box, RAELVNLVG ... KK .... K., beginning at amino acid 60 did not prevent catabolite inactivation. Similarly a change of the vacuole-targeting sequence QKKLD, amino acids 80-84, to QKNSD did not affect significantly the course of inactivation of beta-galactosidase. A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178. A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation. Inactivation of FruP2ase in mutants ubr1 that lack a protein required for ubiquitin-dependent proteolysis, or pra1 that lack vacuolar protease A, proceeded as in a wild type. Our results suggest that at least two domains of FruP2ase may mark beta-galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.