Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 (MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) -148 to -38 and supports binding of numerous transcription factors including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric (dA:dT) element (-119/-110 nt), that conforms to the consensus site for the novel transcription factor Nmp4/CIZ. This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH-responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH-response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5' regulatory region and over-expression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric (dA:dT) element enhances MMP-13 response to PTH and prostaglandin E₂. Over-expression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric (dA:dT) element and the adjacent Runx2 site further augments the PTH response. Based on these data and previous studies, we propose that Nmp4/CIZ is a component of a multi-protein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy.