Chronic liver injury leads to a progressive wound healing response that eventually results in hepatic fibrosis characterised by net deposition of fibrillar extracellular matrix (ECM) and a qualitative shift from type IV to type I/III collagen. The pivotal cellular event underlying this response is hepatic stellate cell (HSC) activation towards a myofibroblast-like phenotype. Activated HSC contribute to ECM remodelling via secretion of type I/III collagens and matrix metalloproteinases (MMPs). Previous studies showed that three-dimensional (3D) contact of activated HSC with type I collagen further stimulates the ECM remodelling properties of HSC by inducing the type IV gelatinase, MMP-9. The aim of the current study was to confirm transcriptional activation of the MMP-9 gene and identify transcription factors regulating this response. Gelatin zymography and Northern blotting were used to confirm induction of MMP-9 protein and mRNA expression in primary rat HSC cultured in a three-dimensional collagen I gel lattice. MMP-9 promoter studies in transfected HSC and electrophoretic mobility shift assay (EMSA) were employed to study transcriptional events. Both NF-kappaB and AP-1 DNA were induced in HSC cultured in 3D collagen I gels and binding sites for these factors in the MMP-9 promoter were crucial for induction of transcription. By contrast removal of an Sp1 site in the promoter enhanced transcription, while over-expression of either Sp1 or Sp3 repressed transcription. It is concluded that 3D contact of activated HSC with collagen I stimulates MMP-9 expression by elevating NF-kappaB and AP-1 activities which are able to overcome the repressive influence of Sp1/Sp3 on MMP-9 gene transcription.