The European Community directive, imposing that effects claimed for cosmetic actives must be validated using non-animal procedures, has stimulated the use of in vitro models for pharmacotoxicological trials. In this paper, an efficacy study of a new biopeptide, a hydrolysate obtained by fermentation of milk proteins, was performed using an in vitro skin equivalent (SE). This SE is obtained by seeding normal human keratinocytes onto a dermal equivalent comprising a collagen-glycosaminoglycan(GAG)-chitosan porous matrix populated by normal human fibroblasts, which neosynthesize their own extracellular matrix (ECM). A gel containing 2% milk biopeptide was applied topically (10 microliters) every 2 days during 15 days. Subsequent investigations of the biopeptide effects were based on morphological criteria after histological analysis and on synthesis of ECM components. Collagen and GAG synthesis were measured by tritiated proline, glucosamine and Na2(35)SO4 incorporation. Qualitatively, the histological features of the biopeptide-treated SEs showed a thicker epidermis than the untreated control SEs, where only a few layers of stratum corneum were observed. The dermal porous matrix seems to be more filled by neosynthesized ECM than the control. Quantitatively, milk biopeptide treatment induced a significant activation of hyaluronic acid (+46%) and sulfated GAG (+53%) synthesis, whereas only non-significant increases of total protein and collagen synthesis were observed (Student's test, p < 0.001).