The pfk3 mutation of Saccharomyces cerevisiae causes glucose-negativity in a pfk1 genetic background, the mutant is temperature-sensitive for growth and homozygous diploids do not sporulate. It fails to accumulate trehalose, and has an altered glycogen accumulation profile under glucose-starvation conditions. pfk3–6, one of the alleles of pfk3, has an altered morphology, forming long chain-like structures at 36°C. The PFK3 gene was cloned by complementation of the mutant phenotypes. Integrative transformation demonstrated that the complementing fragment encoded the authentic PFK3 gene. The disruption of the gene does not affect viability. Like the EMS-induced pfk3 mutant, the disruptants are temperature-sensitive and in a pfk1 genetic background are also glucose-negative. The PFK3 transcript is induced by heat-shock. Partial DNA sequence shows that PFK3 is identical to TPS2 (De Virgilio et al., 1993). We demonstrate that, apart from being a structural determinant of trehalose 6-phosphate phosphatase, PFK3 (TPS2) is required for PFKII synthesis and normal regulation of S. cerevisiae response to nutrient and thermal stresses.