The 92 kd type IV collagenase/gelatinase (MMP-9) is important in mediating basement membrane and extracellular matrix degradation in metastasis. Because MMP-9 is made in tumor cells, but not in quiescent normal cells, we wished to identify the transcriptional elements responsible for its synthesis in tumor cells. We chose to characterize transcriptional regulation of the MMP-9 gene in a highly metastatic H-ras and v-myc transformed rat embryo cell line which overexpresses MMP-9. Using transient transfection of reporter gene constructs containing either 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, we have demonstrated that multiple transcription factor consensus binding motifs in the promoter, including those for NFkappaB, SP-1, Ets, AP-1, and a retinoblastoma binding element, participate in transcriptional regulation of MMP-9 expression in this cell line. Also, deletion of an alternating purine-pyrimidine tract in the downstream promoter was found to decrease transcriptional activity, suggesting that promoter conformation may be important in MMP-9 regulation. Thus multiple pathways leading to activation of NFkappaB, SP-1, Ets, AP-1, and retinoblastoma binding factors in tumor cells all may contribute to MMP-9 transcription and hence to metastasis.