Treatment of the human fibrosarcoma cell line HT-1080 with glucocorticoids results in the induction of fibronectin (FN) protein and mRNA synthesis. We tested the contribution of transcriptional and post-transcriptional mechanisms in the regulation of FN by the synthetic glucocorticoid dexamethasone (DEX). Using nuclear run-on experiments, we found that the DEX-dependent induction of FN occurs primarily at the post-transcriptional level. The half-life of total FN mRNA was not affected by hormone treatment indicating that the induction of FN gene expression is not due to stabilization of the mature message. Interestingly, the induction by DEX was present at the level of nuclear FN RNA. We found that polyadenylation and alternative splicing of the ED-B domain of the FN transcript were not affected by glucocorticoid treatment. However, DEX was found to increase the steady-state level of unspliced FN transcript. Our data indicate that DEX exerts its effect on FN expression predominantly at the post-transcriptional level by a mechanism that, unlike most examples of post-transcriptional regulation by glucocorticoids, acts in the nucleus. Furthermore, they suggest that glucocorticoids activate a mechanism to stabilize the unspliced FN RNA. In an attempt to localize the FN RNA sequences mediating the DEX-dependent induction, we performed transfection analyses of FN minigene constructs. We suggest that the DEX-dependent regulatory elements are located in the introns since no such elements were found in the 8 kb FN mRNA.