Matrix metalloproteinases (MMPs) degrade the extracellular matrix and are implicated in the pathogenesis of several neurological diseases. Secreted in proforms, the MMPs require activation. Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the activity of MMPs. We investigated the expression of MMP-2 and -9, and the role of the TIMP-3 in MMP-2 activation, using cultures of cortical neurons and astrocytes. Under basal conditions, astrocytes and neurons produced low levels of pro-MMP-2, and -9. Stimulation with lipopolysaccharide (LPS) markedly increased pro-MMP-9 production in astrocytes, with only a slight increase in neurons. Pro-MMP-2 were constitutively expressed in both cell types, but with a much higher level in the astrocytes. Real-time RT-PCR showed that the mRNA levels of MMP-2 and -9 paralleled their gelatinolytic activities in the gelatin zymograms. Interestingly, active MMP-2 was observed only in neuronal cultures. TIMP-2 and TIMP-3 are constitutively expressed in astrocytes and neurons. However, astrocytes expressed much higher levels of TIMP-3 mRNA and protein than neurons. Knockdown of TIMP-3 with small interfering RNA (siRNA) significantly increased MMP-2 activation in astrocytes. These results indicate that astrocytes are a more important intrinsic cellular source of MMP-2 and -9 than neurons under normal and neuroinflammatory conditions. TIMP-3 may be the key factor determining the differential activation of MMP-2 in astrocytes and neurons. © 2007 Wiley-Liss, Inc.