The MCP-1 chemokine gene belongs to a cohort of immediate-early genes that are induced with slower kinetics than c-fos. In this study, we identified a cluster of four platelet-derived growth factor (PDGF)-responsive elements within a 240-bp enhancer found in the distal 5' flanking MCP-1 sequences. Two of the elements bind one or more forms of the transcription factor NF-kappa B. We focused on the other two elements which are hitherto unreported, PDGF-regulated genomic motifs. One of these novel elements, detected as a 28-mer by DNase I footprinting, restores PDGF inducibility when added in two copies to a 5' truncated MCP-1 gene. A single copy of the second novel element, a 27-mer, restores PDGF inducibility to a 5' truncated MCP-1 gene. The 27-base element interacts with a PDGF-activated serine/threonine phosphoprotein that is detected only within the nucleus of PDGF-treated 3T3 cells. DNA binding of this phosphoprotein is activated by PDGF treatment with slow kinetics that match the time course of MCP-1 gene expression, and activation is not inhibited by cycloheximide. PDGF-activated binding to the 27-mer is shown to involve a single 30-kDa protein by UV-cross-linking analysis.