Hypoxia not only controls organogenesis, embryogenesis, and wound repair, but also triggers tumor progression and metastasis. Matrix metalloproteinases (MMP), especially gelatinases (MMP‐2, MMP‐9) regulate the composition and stability of the extracellular matrix (ECM), which affects cell proliferation, migration, and differentiation. This study investigated the effect of hypoxia alone and in combination with ECM compounds and nutrition on MMP‐2 and MMP‐9 expression, activity, and synthesis in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMC). We also determined the expression of the tissue inhibitors of MMP (TIMP‐1, ‐2). Cells were grown on plastic, collagen‐I, collagen‐IV, or gelatin and in either starving medium (0.1% serum) or growth medium (5% serum), and were subjected to normoxia or hypoxia (1% O₂). Collagenases expression was determined by zymography. TIMP‐1, ‐2 expression was assessed by Western blotting and RT‐PCR. Depending on serum concentration human lung cells expressed pro‐MMP‐2 on all substrates. Hypoxia increased pro‐MMP‐2 expression, on collagen type I or type IV further via Erk1/2 and p38 MAP kinase signaling. MMP‐9 was only expressed when cells were grown on collagen type IV and increased with serum concentration, and by hypoxia. TIMP‐1 expression was only expressed when cells were grown on collagen type I and was significantly increased by hypoxia, while TIMP‐2 expression was unchanged. We demonstrated that the hypoxia, ECM composition, and nutrition, rather than one of these conditions alone, modulate the expression and activity of collagenases and their inhibitors in primary human lung fibroblasts. © 2005 Wiley‐Liss, Inc.