Human kcratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squanious epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-12 cells can partially substitute for the intact feederlayer, we studied the possible involvement of insulin-like growth lactors acting in a paracrine fashion. IGFs were measured (after Scphadex G-50 gel-chromatography in acid conditions) in media conditioncd by a feeder-layer of lethally irradiated 3T3-12 fibroblasts on which keratinocytes were grown. lmmunoreactive (IK) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-l and ICF-II, and conditioned medium from 3T3-12 cells, caused a dose-dependent increase of 'H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-)2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alK-3, which is a specific antagonist of type-l IGF receptor. Fetal mouse-derived 3T3-)2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition oiSm 1.2 MoAb causes a 50?A decrease in the keratinocyte number. When the non-IGF-producing BALBic 3T3 cells were used as a feedcrlayer, the keratinocytes number was similar to that observed with JT3-JZ and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-12 and of norrnal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.