MT3-MMP is a matrix metalloproteinase involved in the regulation of cancer cell invasion and metastasis. The MT3-MMP mRNA contains a 20-nucleotide G-rich region (M3Q) upstream of the initiation codon. Herein, we report that the M3Q purine-only sequence forms an extremely stable intramolecular G-quadruplex structure and has an inhibitory role on translation of a reporter gene in eukaryotic cells. The formation of the G-quadruplex structure was indicated by circular dichroism (CD) spectroscopy and enzymatic footprinting with RNase T1. The unusual stability of the G-quadruplex was evidenced when addition of only 1 mM KCl resulted in about a 30 degrees C increase in the melting temperature (T(m)), as compared to that obtained in the absence of added salt. The T(m) was independent of the RNA concentration, suggesting an intramolecular G-quadruplex structure. Additionally, in a dual luciferase reporter assay performed in eukaryotic cells, the M3Q motif present in the context of the entire 5'-UTR of MT3-MMP repressed activity of its downstream gene by more than half. To the best of our knowledge, the naturally occurring M3Q sequence forms one of the most stable, intramolecular RNA G-quadruplexes reported. This report is the first to establish a functional role of a G-quadruplex forming sequence within the MT3-MMP 5'-UTR in the regulation of translation in eukaryotic cells.