Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly. Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms. Using peptide sequences from the erythroid 68-kDa PIP5KI, we have isolated cDNAs encoding PIP5KIalpha from human brain. Partial cDNAs obtained for a second isoform, PIP5KIbeta, established that the human STM7 gene encoded a previously unrecognized PIP5KI. However, the peptide sequences demonstrated that erythroid PIP5KI corresponded to PIP5KIalpha. Recombinant, bacterially expressed PIP5KIalpha possessed PIP5K activity and was immunoreactive with erythroid PIP5KI antibodies. By Northern analysis, PIP5KIalpha and PIP5KIbeta had wide tissue distributions, but their expression levels differed greatly. PIP5KIs had homology to the kinase domains of PIP5KIIalpha, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base. These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes. Furthermore, the limited homology between PIP5KIs and PIP5KIIalpha, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks.