Transcriptional status of the genes needed for galactose utilization in Saccharomyces cerevisiae is controlled by the interplay between the prototypical transcriptional activator Gal4p and the inhibitor protein Gal80p. Relief of the inhibition from Gal4p requires the interaction between Gal80p and the galactokinase paralog, Gal3p. Here, we present evidence that decrease in the intracellular levels of ATP or NADP(H) impairs the GAL gene expression. All these induction defects are rescued by overproducing Gal3p or producing Gal4p mutants with reduced interaction with Gal80p. We further demonstrate that removal of Gal80p from the GAL gene promoter is impaired in these mutants, and that NADP(H) cooperates with Gal3p in causing the dissociation of Gal80p from the in vitro preformed DNA-bound Gal80p–Gal4p complex. We also show that Gal80p is only partially removed from the GAL gene promoter in a mitochondria fusion-deficient mutant where the cotranscriptional mRNA processing is crippled. The efficient dissociation is restored by Gal4p mutants with altered interaction with Gal80p and is correlated with the recovered GAL gene expression. These results indicate that multiple metabolic signals exist to facilitate the efficient and appropriate dissociation of Gal80p from Gal4p by Gal3p to achieve the fully active state of Gal4p in the nucleus.