The purpose of this study was to evaluate the influence of fibrin glue and aprotinin on the growth of adult human skin keratinocytes in defined serum-free conditions. The keratinocytes were cultured on cell culture plastics and on a fibrin matrix prepared from fibrin glue. The cell growth was measured by MTT assay, while the growth of clonogenic keratinocytes was evaluated by colony assay and expressed as colony-forming efficiency (CFE). The clonogenic potential of keratinocytes released from subconfluent and confluent cultures grown on fibrin glue was also studied by the colony assay. In comparison to a plastic culture surface the fibrin glue had significantly (P<0.05) increased the clonogenic potential of keratinocytes, as well as enhanced their growth. Keratinocytes released from subconfluent cultures grown on fibrin glue attained a significantly (P<0.05) higher percentage of clonogenic cells than their confluent parallels. At 75, 150, 300 and 450 KIU/ml aprotinin did not influence the growth of keratinocytes (P>0.2). A fibrin-based skin substitute produced in the defined keratinocyte medium could be safely used to treat a number of skin defects.