Studies in rodents demonstrate that the mitogen, insulin-like growth factor I (IGF-I), stimulates intestinal peptide YY (PYY) expression. To investigate whether the stimulatory influence of IGF-I is exerted at the level of gene transcription, rat PYY 5' -upstream sequences (-2800/+37 bp, -770/+37 bp, -127/+37 bp) fused to the firefly luciferase reporter gene were transfected into rat pheochromocytoma cells (PC12) and luciferase activity measured following IGF-I treatment. IGF-I increased transcriptional activity of all constructs similarly; the PYY (-127/+37 bp)-luciferase (luc) construct was used in subsequent experiments. IGF-I increased PYY (-127/+37 bp)-luc activity in a time- and dose-dependent fashion. Sequence analysis detected 5 putative Sp1 binding sites in the -127/+37 bp sequence. EMSA and supershift experiments using two oligonucleotide fragments of the -127/+37 region showed that Sp1 and Sp3 proteins bound to putative Sp1 sites. Over-expression of Sp1 greatly increased PYY (-127/+37 bp)-luc activity and site-directed mutagenesis of putative Sp1 binding sites decreased basal and IGF-I-induced elevations in PYY (-127/+37 bp)-luc activity. IGF-I treatment also increased Sp1 protein levels and binding activty. Blockade of the IGF-I receptor (IGF-IR) with an IGF-IR antibody decreased the stimulatory influence of IGF-I on Sp1 protein levels and PYY (-127/+37 bp)-luc activity. Together, these findings indicate that IGF-I functions as a positive regulator of PYY gene expression and that the stimulatory effect may be mediated by Sp1 proteins that bind to the proximal PYY promoter region.