Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. The PAI-2 promoter tightly regulates PAI-2 gene expression in a cell-specific manner and this control is mediated, in part, by the upstream silencer element, PAUSE-1. Here we have defined PAUSE-1 and investigated its activity as a silencer. A series of mutations were generated within the PAUSE-1 element and analysed for transcription factor binding and transcriptional silencing activity. These studies have defined the minimal functional PAUSE-1 element as TCTN(x)AGAN(3)T(4), where x = 0, 2 or 4. Examination of related elements present in other promoters, such as the human IFNbeta promoter, suggests that PAUSE-1 is a member of a family of universal silencers with the consensus sequence TCTN(x)AGA. UV crosslinking analyses determined that the PAUSE-1 binding protein was approximately 67 kDa. Insertion of PAUSE-1 into the heterologous (SV40) or the minimal PAI-2 promoters silenced transcription by 2.5-fold. These data show that PAUSE-1 acts as a powerful silencer of PAI-2 gene transcription and is likely to be important in the silencing of other genes as well.