In cultured murine fibroblasts, the TIMP gene (encoding tissue inhibitor of metalloproteinases) is transcribed constitutively, although at low levels. We have used a cell-free system in which nuclear extracts prepared from murine L cells support transcription from TIMP DNA templates in vitro. This system was used to study the role of cis-acting DNA sequences in the constitutive expression of TIMP. Sequences important for expression are located both 5' and 3' (in intron 1) to the major transcription start point and are required to obtain detectable levels of transcription. These regions are specifically recognized by murine nuclear factors and contain DNA motifs whose sequences closely resemble binding sites for known transcriptional activators. In particular, the data strongly suggest a role for CCAAT-binding factor(s) and AP1-binding factors in the basal transcription of TIMP.