The production of the thymic hormone, thymulin (FTS), was studied in primary cultures of human thymic epithelium by immunofluorescence using monoclonal anti-thymulin antibodies. The number of thymulin-containing cells and the thymulin level in the culture supernatant increased gradually during the culture. Addition of synthetic thymulin to the culture medium reduced significantly the increase of thymulin-containing cells. Conversely, addition of monoclonal anti-thymulin antibody from the beginning of the culture exacerbated the spontaneous increase of thymulin-containing cells and abrogated the effects of thymulin. Combined with similar data previously reported in vivo, these results demonstrate that thymulin is actively produced by cultured thymic epithelial cells and that its synthesis can be down-regulated by the hormone itself.