Increased airway smooth muscle (ASM) mass and infiltration by mast cells are key features of airway remodeling in asthma. We describe a model to investigate the relationship between ASM, the extracellular matrix, mast cells, and airway remodeling. ASM cells were cultured in a three-dimensional (3-D) collagen I gel (3-D culture) alone or with mast cells. Immunocytochemistry and Western blotting of ASM in 3-D cultures revealed a spindle-shaped morphology and significantly lower alpha-smooth muscle actin and vimentin expression than in ASM cultured in monolayers on collagen type I or plastic (2-D culture). In 3-D cultures, basal ASM proliferation, examined by Ki67 immunocytochemistry, was reduced to 33 +/- 7% (P < 0.05) of that in 2-D cultures. The presence of mast cells in cocultures increased ASM proliferation by 1.8-fold (P < 0.05). Gelatin zymography revealed more active matrix metalloproteinase (MMP)-2 in 3-D than in 2-D culture supernatants over 7 days. Functional MMP activity was examined by gel contraction. The spontaneous gel contraction over 7 days was significantly inhibited by the MMP inhibitor ilomastat. Mast cell coculture enhanced ASM gel contraction by 22 +/- 16% (not significant). Our model shows that ASM has different morphology, with lower contractile protein expression and basal proliferation in 3-D culture. Compared with standard techniques, ASM synthetic function, as shown by MMP production and activity, is sustained over longer periods. The presence of mast cells in the 3-D model enhanced ASM proliferation and MMP production. Airway remodeling in asthma may be more accurately modeled by our system than by standard culture systems.