Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2 strain also exhibits a severe imbalance in intracellular Ca homeostasis when grown under these conditions. In the present study, we examined how the pgm2 mutation alters yeast Ca homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca uptake in wild-type cells, whereas Ca uptake increased 8-fold in the pgm2 mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca -ATPase Pmc1p, suppressed the Ca -related phenotypes observed in the pgm2 strain. This suggests that excessive vacuolar Ca uptake is tightly coupled to these defects in Ca homeostasis. An in vitro assay designed to measure Ca sequestration into intracellular compartments confirmed that the pgm2 mutant contained a higher level of Pmc1p-dependent Ca transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca uptake also coincided with a large induction of the unfolded protein response in the pgm2 mutant, suggesting that Ca uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca uptake and accumulation previously shown to be associated with the pgm2 mutation are due to a severe imbalance in the distribution of cellular Ca into different intracellular compartments.