Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi-layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-beta1 (TGF-beta1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms express different levels of TGF-beta1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 microg/ml) and EGF (5 microg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi-layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF-beta1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and (3)H-thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF-beta1, involucrin, TIMP-1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1-2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF-beta1 evaluation revealed that mono-layers of cultured keratinocytes which were round, attached, and proliferating in KSFM + BPE and EGF containing medium released a significantly higher level of TGF-beta1 (196 +/- 58 pg /ml) relative to those grown in multi-layer forms (28 +/- 7.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF-beta1 (388 +/- 53 pg/ml) relative to those grown in KSFM + BPE and EGF. This finding was consistent with the expression of TGF-beta1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF-beta1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of (3)H-thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF-beta1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF-beta1 and a fibroblast mitogenic factor.