Several in vivo experimental and clinical studies suggest that the production of thymic hormones, such as thymulin (Zn-FTS), is modulated by thyroid hormones. It was not determined in these studies, however whether such modulation is exerted directly on the thymic epithelial cells which synthesize and secrete thymic hormones. In order to discriminate between direct and indirect modulation, the effect of thyroid hormones on the in vitro production of thymulin by whole thymic organ culture, as detected by the rosette inhibition assay, has been investigated. Donors of thymuses were young 6N-propyl-2 thiouracil (PTU)-treated hypothyroid Balb/c mice and normal littermates. Thymuses from hypothyroid mice were shown to produce concentrations in vitro nearly undetectable of thymic hormone, when compared to thymuses from normal mice. The in vitro addition of triiodothyronine (T3) caused a complete recovery of the thymic hormone production by thymuses from hypothyroid mice and an increased synthesis even by normal thymuses over control values. The complete blockade of in vitro thymic hormone production with cycloheximide, which inhibits mRNA and protein synthesis but not thyroid hormone permissive actions, suggests that the T3 induced increment of thymic hormone level in the supernatant is due to de novo synthesis. Furthermore, the number of thymulin-producing cells, as detected by immunofluorescence using a specific antithymulin monoclonal antibody, which is quite low in thymuses from hypothyroid mice, is completely regained after in vitro incubation with T3. These findings support the idea that the modulation of thyroid hormones on thymic endocrine activity is directly exerted at thymic level.