The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFα or anti-IL-1β-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1β, TNFα, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFα, and IL-1β. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFα neutralizing antibody but not with anti-IL-1β. No significant effect of LPS, TNFα, or IL-1β on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFα-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFα appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFα and IL-1β produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.