Three-dimensional micropatterned collagen scaffolds were fabricated by femtosecond laser ablation. An 800 nm, 45 fs Ti:Sapphire laser was used to create various 3D patterns in a collagen gel, including holes, lines and grids. An optimal collagen concentration was found for both substrate patterning and cell compatibility. The threshold fluence for ablation of the scaffold was found to be 0.0 6 J/cm(2), and the morphology of the ablation craters was measured as a function of fluence. Mesenchymal stem cells from rat bone marrow and human fibroblasts were seeded within the ablated patterns and were shown to be viable for at least 10 days.