Although the intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at a low level on a subpopulation of hematopoietic cells, on vascular endothelium, on fibroblasts, and on certain epithelial cells, it is dramatically increased at sites of inflammation. Interferon-gamma (IFN-gamma) and phorbol myristate acetate (PMA) are known to increase the expression of ICAM-1 on many cell types. Because both human and murine ICAM-1 mRNAs contain putative destabilizing AUUUA sequences in their 3′ untranslated regions (UTRs), we examined the role of mRNA stability in the regulation of ICAM-1 gene expression. The treatment of the murine monocytic cell line P388D1, which constitutively expresses ICAM-1 mRNA at a low level, with IFN- gamma or PMA rapidly enhanced the level of ICAM-1 mRNA and dramatically prolonged its half-life. To determine whether the putative destabilizing sequences are responsible for this effect of IFN-gamma and PMA, fibroblast L cells were transfected with either the full- length ICAM-1 cDNA or a truncated form (ICAM-1 delta 3) lacking the putative destabilizing AUUUA sequences. Although ICAM-1 delta 3 mRNA was more stable than the full-length ICAM-1 mRNA, IFN-gamma treatment induced the accumulation of both mRNA species and prolongation of their half-lives. The transplantation of the ICAM-1 delta 3′ UTR into a stable ICAM-2 mRNA rendered it unstable, and it was unresponsive to IFN- gamma. Therefore, the treatment with IFN-gamma stabilizes the otherwise labile ICAM-1 mRNA, but the IFN-gamma-responsive sequence may at least in part reside within the protein coding region. PMA also upregulated ICAM-1 gene expression by mRNA stabilization. However, unlike IFN- gamma, PMA treatment only increased the level of the full-length, but not of the truncated, ICAM-1 mRNA. This shows that the PMA-responsive element is located within the 3′UTR. Furthermore, the effect of PMA on ICAM-1 delta 3 mRNA was recovered by ligating multiple AUUUA sequences derived from a heterologous gene fragment. The stability of this chimeric mRNA and the full-length ICAM-1 mRNA was markedly increased by PMA treatment, indicating that the AUUUA multimers in the 3′UTR are important in the PMA-induced upregulation of ICAM-1 mRNA.