Cultured human epidermal cells have been shown to produce soluble factors endowed with T cell differentiating activities. In addition, the presence of thymopoietin and FTS/thymulin-like factors has been reported in normal human and mouse epidermis using immunohistochemical methods and anti-thymic hormone antibodies. The present study was conducted to re-evaluate the presence of thymic hormones in normal epidermal cells using a panel of monoclonal antibodies (mAbs) and rabbit antisera to several well characterized thymic factors. The reactivity of the following antibodies was tested by indirect immunofluorescence on human and mouse tissue sections: a) two anti-FTS/thymulin mAbs; b) one anti-FTS/thymulin rabbit antiserum; c) one anti-thymopoietin rabbit antiserum; d) one anti-thymosin alpha 1 mAb. Our results show that: 1) all five antibodies reacted with human and/or mouse thymic epithelial cells; 2) none of the 3 antithymic hormone mAbs (2 anti-FTS/thymulin and 1 anti-thymosin alpha 1 mAbs) reacted with normal skin; 3) only 2 out of 5 antibodies, namely the anti-FTS and anti-thymopoietin antisera cross-reacted with mouse and human epidermis and labeled keratinocytes, as previously reported; these latter 2 antibodies also stained nude mouse epidermal cells and labeled non-thymic, non-epidermal normal mouse epithelial tissues, suggesting that the cross-reactive epitope is common to a number of epithelial cells; 4) the antigen defined by the anti-FTS and anti-thymopoietin antisera was not related to keratins, since absorption experiments using purified human epidermal keratins failed to abolish staining of the epidermis. We conclude from this study that epidermal cells do not produce in vivo the well characterized thymic hormones: FTS/thymulin, thymopoietin and thymosin alpha 1. The precise nature of the antigenic structure recognized within epidermis by the anti-FTS and anti-thymopoietin antibodies remains to be defined.