Thymulin (or serum thymic factor, FTS-Zn), a well-defined thymic hormone previously shown to be a nonapeptide binding the metal zinc, was studied in mice subjected to a long-term marginally Zn-deficient diet. In spite of the absence of thymic atrophy, we observed a significant decrease in the serum levels of thymulin as early as two months after the onset of treatment. However, these levels could be consistently restored after in vitro addition of ZnCl2. The analysis of thymuses from Zn-deficient mice showed that, despite the apparently normal network of epithelial cells, there was a progressive increase in the number of thymulin -containing cells (assessed by immunofluorescence with anti- thymulin monoclonal antibodies) that was already significant after two months of treatment. These results are in keeping with those of previous investigators, showing a specific, altered, thymic endocrine function following Zn deprivation. Nonetheless, our results strongly suggest that the nonactive Zn-deprived peptide is secreted under these experimental conditions. Furthermore, the fact that the augmented numbers of thymulin -containing cells were observed in the thymuses following a decrease in the peripheral thymulin (biologically active) brings further evidence for the existence of a feedback mechanism for the secretion of this hormone.