We have recently demonstrated that interleukin-1β (IL-1β) stimulates matrix metalloproteinase-9 (MMP-9) induction. In this study we have investigated the roles of superoxide and extracellular signal-regulated kinase (ERK) activation in MMP-9 induction following exposure to IL-1β. IL-1β stimulated biphasic ERK activation in vascular smooth muscle (VSM) cells, a transient activation that reached a maximum at 15 min and declined to baseline levels within 1 h, and a second phase of sustained ERK activation lasting up to 8 h. To determine the role of ERK in IL-1β-stimulated MMP-9 induction, we treated cells with the specific ERK pathway inhibitor PD-98059 at different time intervals after IL-1β stimulation. Addition of PD-98059 up to 4 h after IL-1β stimulation significantly inhibited MMP-9 induction, suggesting a role for sustained ERK activation in MMP-9 induction. IL-1β treatment stimulated superoxide production in VSM cells that was inhibited by pretreatment of cells with the superoxide scavengerN-acetyl-l-cysteine (NAC) and also by overexpression of the human manganese superoxide dismutase (MnSOD) gene. Treatment of VSM cells with NAC selectively inhibited the sustained phase of ERK activation without influencing the transient phase, suggesting a role for reactive oxygen species in sustained ERK activation. In addition, both NAC treatment and MnSOD overexpression significantly inhibited IL-1β-stimulated MMP-9 induction (P < 0.05). The results demonstrate that IL-1β-dependent MMP-9 induction is mediated by superoxide-stimulated ERK activation.