BACKGROUND Cultured dermal substitutes are used for the treatment of skin ulcers. However, the biological risks of fetal bovine serum (FBS) in the culture process have been reported. The use of the patient's autologous serum (AS) is another possibility, but the amount available is limited. In this study, we examined whether animal product-free media (HFDM-1) supplemented with 2% AS could support the growth of autologous fibroblasts in primary culture and their dissemination to dermal substitutes. METHODS We cultured autologous fibroblasts using HFDM-1 with 2% AS, Dulbecco's modified eagle medium (DMEM) with 10% FBS, and DMEM with 10% human serum (HS). Then, we disseminated and cultured the cells for 10 d. The fibroblast proliferation and concentrations of vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) in each medium, as well as the deposition of human type I collagen into dermal substitutes were examined. RESULTS The number of fibroblasts cultured in HFDM-1 with AS was highest. After seeding, the number of fibroblasts cultured in DMEM with HS was higher than those in DMEM with FBS and HFDM-1 with AS, but no significant difference was found between these two media. The VEGF concentration in DMEM with HS was also larger, but no significant difference was found between two other media. No significant difference was observed in TGF-β1 concentration or the deposition of collagen. CONCLUSIONS This study shows that HFDM-1 with 2% AS can be used to produce cultured dermal substitutes instead of DMEM with 10% FBS.