Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first committed step in the biosynthesis of phosphatidylinositol 4,5-bisphosphate. Here we report the first mammalian cDNA clone of a PtdIns 4-kinase (named PI4K alpha). The 2.6-kb cDNA encodes a protein of 854 amino acids that is highly homologous to the recently cloned yeast PtdIns 4-kinase STT4 and is also homologous to a second yeast PtdIns 4-kinase, PIK1. PI4K alpha has more distant sequence homology to the catalytic domains of mammalian and yeast PtdIns 3-kinases and to the yeast Tor family of proteins. It also has a region of similarity to pleckstrin homology domains and a potential ankyrin repeat. Cross-hybridizing messages were detected in all human tissues investigated. The enzymatic properties of the protein expressed in insect cells are characteristic of type II PtdIns 4-kinases (activated by detergent and inhibited by adenosine), and PI4K alpha is recognized by an antibody specific for type II PtdIns 4-kinases.