Transmission of mitogenic and developmental signals to intracellular targets is often mediated by inositol derivatives. Here we present the cloning and characterization of a gene from Saccharomyces cerevisiae, PIK1, encoding the enzyme that catalyses the first committed step in the production of the second messenger inositol-1,4,5-trisphosphate. PIK1 encodes a phosphatidylinositol 4-kinase (PI 4-kinase) essential for growth. Cells carrying PIK1 on a multicopy vector overexpress PI 4-kinase activity exclusively in a nuclear fraction, suggesting that PIK1 is part of a nuclear phosphoinositide cycle. Temperature-sensitive mutations, but not a null mutation, can be suppressed by high osmolarity or an elevated concentration of Ca2+. Conditional mutants have a cytokinesis defect as indicated by a uniform terminal phenotype of cells with large buds and fully divided nuclei. We suggest that PIK1 controls cytokinesis through the actin cytoskeleton.