The engineering of dermal skin substitutes, using autologous fibroblasts, requires high seeding efficiencies, a homogeneous cell distribution in the scaffolds, and optimal culture conditions. Dynamic seeding in spinner flasks was used to seed and subsequently culture fibroblasts in three-dimensional scaffolds. Several seeding and culture variables were investigated. Simulation of medium movement with microspheres showed that three different regions existed in medium (outer, middle, and inner), where overall particle movement was different. In the middle region the flow was turbulent and scaffolds were best placed in this region. After fibroblast seeding, methylene blue staining and scanning electron microscopy analysis of the scaffolds showed that at a low stirring speed (20 rpm) fibroblasts attached mainly onto the upper part of the scaffold, and at 40 and 60 rpm fibroblasts attached and spread throughout the scaffolds. Measurements of total DNA content per scaffold showed that lower stirring speeds (20 and 40 rpm) resulted in significantly higher cell-seeding efficiencies (20 rpm, 99.8 ± 11.3%; 40 rpm, 93.8 ± 10.5%) compared with 60 rpm (85.9 ± 5.3%). Seeding kinetics were comparable for all three speeds investigated. In subsequent studies, 40 rpm was chosen for seeding. Using initial cell numbers ranging from 0.3 × 106 to 1.5 × 106 fibroblasts per scaffold, seeding efficiencies higher than 85% were consistently found (n = 4). The culture of fibroblast-seeded scaffolds at different stirring speeds (10-80 rpm) showed that stirring speeds higher than 10 rpm significantly stimulated fibroblast proliferation and glycosaminoglycan and collagen deposition as compared with 10 rpm. After 21 days, scaffolds cultured at 80 rpm showed significantly more collagen deposition as compared with those maintained at lower speeds. In conclusion, to achieve high seeding efficiencies, uniform fibroblast distribution and tissue formation in a three-dimensional scaffold, fibroblasts can be dynamically seeded at 40 rpm and subsequently cultured at a stirring speed of 60-80 rpm in spinner flasks. This flexible system shows that it is feasible to tissue engineer autologous dermal substitutes in a clinically acceptable time frame.