Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of Ptdlns-4,5-bisphosphate (PtdIns[4,5]P sub 2). Hydrolysis of Ptdlns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that modulate cell proliferation and differentiation. The PIK1 gene encoding a Ptdlns 4-kinase from the yeast Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast (Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.