The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.