T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.