In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.