Type I collagen was prepared from rat tail tendons as described (Elsdale and Bard 1972) and dissolved in 0.2% acetic acid to a final concentration of 2.7 mg/ml. To induce gelling, collagen was mixed with 10× DME and 0.34 N NaOH in an 8:1:1 ratio at 4°C, and 1 ml of this mixture was added to the upper well of a 24-mm Transwell dish (3-μm pore size; Corning, Inc.). After gelling was complete (45 min at 37°C), 2–5 × 105 cells in complete medium were added to the upper well. After an additional 24-h incubation period, SF/HGF was added to the lower compartment of the Transwell chambers at a final concentration of 50 ng/ml. In indicated experiments, protease inhibitors were added to both the upper and lower wells at the following final concentrations: 5 μM BB-94 (0.1% DMSO; final concentration); 200 ng/ml recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1; Oncogene Research Products); 200 ng/ml recombinant TIMP-2 (gift of Amgen, Thousand Oaks, CA); 200 μg/ml aprotinin; 10 μM bestatin; 100 μg/ml soybean trypsin inhibitor (SBTI); 100 μM E64 (0.1% ethanol; final concentration); and 50 μM pepstatin (0.1% methanol; final concentration; all from Sigma-Aldrich). None of the solvents used affected MDCK or COS-1 cell behavior when tested alone. FCS was depleted of plasminogen or gelatinase, respectively, by lysine-sepharose or gelatin-sepharose (both from Amersham Pharmacia Biotech) affinity chromatography (Rosenthal et al. 1998). All media, including SF/HGF and inhibitors, were replaced every 2 d. MDCK and COS-1 invasion assays were routinely terminated after 12 and 5 d, respectively. Invasive foci were counted in randomly selected fields at 20× on a phase-contrast microscope. Invasion depths were measured from digitally captured images of hematoxylin and eosin-stained cross-sections.