Cyclooxygenase-2 (COX-2) is a highly inducible gene in macrophages by pro-inflammatory cytokines. A major mechanism for cytokine-induced COX-2 expression is stabilization of COX-2 mRNA. In this study, we examined the induction of COX-2 expression by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in human primary in vitro differentiated macrophages. IL-1β (5 ng/mL) or TNF-α (1 ng/mL) induced up to an ∼40-fold increase of COX-2 mRNA in macrophages during a 2 to 2.5-hr incubation. Run-off experiments demonstrated that cytokine stimulation had only a mild effect on the COX-2 transcription rate (∼10–40% increase). The translation blocker cycloheximide (CHM) (10 mg/mL) superinduced COX-2 mRNA during 2 hr of incubation and further stabilized the COX-2 mRNA (T_1/2 > 4 hr). The CHM-superinduced COX-2 mRNA was subject to a rapid degradation after removal of CHM (T_1/2 < 1 hr). Both IL-1β and TNF-α stabilized cytokine-induced COX-2 mRNA (T_1/2 ≥ 2 hr). Maximal stabilization of COX-2 mRNA after a short-term stimulation required the continued presence of IL-1β in the medium. Long-term treatment of TNF-α destabilized the induced COX-2 mRNA. Cells simultaneously treated with both IL-1β and TNF-α had a reduced induction of COX-2, IL-1β, and IL-6 mRNA. In transcription-arrested cells, the translation blocker puromycin affected the TNF-α-induced stabilization and destabilization of COX-2 mRNA, but not the IL-1β-induced stabilization. The studies suggest that positive and negative regulation of mRNA stability may play a major role in cytokine-mediated COX-2 induction in human macrophages. TNF-α may play both pro-inflammatory and protective roles during inflammation by regulation of pro-inflammatory gene transcripts.