Glucocorticoids are potent anti-inflammatory agents widely used in the treatment of human disease. We have previously shown that the inflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) is regulated posttranscriptionally by glucocorticoids in arterial smooth muscle cells (SMC). To elucidate the mechanism mediating this effect, in vitro-transcribed radiolabeled MCP-1 mRNA was incubated with cytoplasmic extracts from SMC and analyzed by gel electrophoresis. Extracts from SMC treated with platelet-derived growth factor (PDGF) did not degrade the transcripts for up to 3 h. In contrast, extracts from cells treated with 1 μM dexamethasone (Dex) alone or in combination with PDGF degraded the probe with a half-life of ≈15 min. Dex had maximal effect at concentrations above 0.01 μM and was effective on both rat and human MCP-1 transcripts. By deletion analysis, the Dex-sensitive region of the MCP-1 mRNA was localized to the initial 224 nucleotides (nt) at the 5′ end and did not involve an AU-rich sequence in the 3′ untranslated end. The 224-nt region conferred Dex sensitivity to heterologous mRNA. These studies provide new insights into the molecular mechanisms underlying the effect of glucocorticoids on gene expression.