Keratinocytes are frequently used to examine efficacy of wound healing products and dermatological agents in vitro. Cultured keratinocyte sheets are also used as autologous or allogenic grafts to promote wound closure. Because it is well known that the expression patterns of keratin genes change when cell cultures reach confluence, we investigated the expression pattern of wound healing-related genes, including growth factors and cytokines. Of additional particular interest is a novel wound healing related factor, secretory leukocyte protease inhibitor (SLPI), which appears to enhance tissue repair. We found that the expression pattern varied for specific genes expressed by keratinocytes as confluence was reached. Specifically, SLPI expression peaked in the early postconfluent state and vascular endothelial growth factor and amphiregulin in the late postconfluent state. Some gene products exhibit autocrine activity, whereas others exert paracrine regulation of growth. These findings indicate that it is critical to define the growth and differentiation state of human keratinocyte cultures to better determine responses and efficacy in vitro to various dermatological/wound care agents tested.