While matrix stiffness has been implicated in cell adhesion and migration, most studies have focused on the effects of substrate stiffness in 2D. The present work describes a novel continuous stiffness gradient model for studying such processes in 3D. Wedge-shaped collagen scaffolds were compressed to produce sheets of a desired (0.1 mm) uniform thickness, but with increasing collagen density along the length of the sheet. Dynamic mechanical analysis, carried out on 1 mm wide strips obtained from the two ends and the middle of each sheet, showed that the elastic modulus increased from 1057 ± 487 kPa to 2305 ± 693 kPa at the soft and stiff end respectively and was 1835 ± 31 kPa in the middle. In constructs seeded with agarose marker beads prior to compression, mean agarose bead density rose from 10 ± 1 to 71 ± 12 at the soft and stiff end respectively and was 19 ± 5 in the middle, indicating successful engineering of a density gradient corresponding to the measured stiffness gradient. Growth-arrested human dermal fibroblasts, initially seeded evenly within such constructs, accumulated preferentially towards the stiff part of the gradient after 3 and 6 days in culture. Durotactic migration was significant after 6 days. This model provides a new means for studying cellular mechanotaxis and patterning cells which is controllable, biomimetic and in 3D. Cell Motil. Cytoskeleton 66: 121–128, 2009. © 2009 Wiley-Liss, Inc.