We have isolated a new mutant of Saccharomyces cerevisiae that exhibits a glucose-derepression resistant (and sucrose-non-fermentor) phenotype. This mutant was obtained by screening for overproduction of alpha-amylase in a strain containing the mouse alpha-amylase gene under the control of the PGK promoter. The mutation designated pop2 (PGK promoter directed over production). The pop2 mutant overproduced amylase 5-6 fold and displayed several other pleiotropic defects: (1) resistance to glucose derepression, (2) temperature-sensitive growth, (3) failure of homozygous diploid cells to sporulate and (4) reduced amount of reserve carbohydrates. We mapped pop2 to chromosome XIV, distal to lys9 and SUP28, indicating that POP2 is a newly-identified locus. We isolated the POP2 gene from two yeast strains of different genetic backgrounds, S288C and A364A, and determined their nucleotide sequences. The predicted amino acid sequence of the POP2 protein contains three glutamine-rich region, a proline-rich region and a serine/threonine-rich region, characteristic of many transcription factors. Steady state levels of RNA transcribed from the PGK-amylase fusion gene and from endogenous PGK gene in stationary-phase pop2 cells were 5- to 10-fold higher than those observed in wild-type cells, showing that the pop2 mutation affects transcription of the PGK gene transcription.