Like other nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) use a wide variety of protein-protein interactions to properly regulate transcription of target genes. In an attempt to identify novel PPAR-interacting proteins, a cDNA expression library was screened with bacterially expressed PPARalpha. One of the genes identified as a PPARalpha-associated protein by interaction cloning was the CREB-binding protein/p300-interacting transactivator with ED-rich tail 2 (CITED2, also called p35srj/mrg1/msg1). This coactivator interacted directly with PPARalpha in the presence or absence of ligand predominantly via the ligand binding domain of the nuclear receptor. In transient transfection reporter assays, CITED2 acted as a dose-dependent coactivator of PPARalpha-dependent transcriptional regulation in the presence of several exogenous ligands. CITED2 also increased PPARgamma-dependent regulation of reporter genes but had no effect on PPARbeta activity. To determine whether CITED2 affects endogenous gene expression, this protein was stably overexpressed (CITED2+) or repressed by small inhibitor RNA (CITED2-) in immortalized mouse hepatocytes. Relative to the control stably transfected or CITED2-cells, CITED2+ cells had an increased rate of cell proliferation. Microarray analysis and real time PCR showed that several genes are differentially affected by PPARalpha ligands in CITED2+ versus CITED2-cells. Genes that were affected by PPARalpha ligands in a CITED2-modulatory manner include angiopoietin-like protein 4, forkhead C2, hypoxia-inducible factor-1alpha, and MAPK phosphatase 1. Interestingly these genes share common functions in that they are known to promote vascularization and angiogenesis in response to hypoxia. The results described here suggest that CIT-ED2 is a coactivator of PPARalpha and that both proteins may participate in signaling cascades of hypoxic response and angiogenesis.