We have recently cloned the promoter of the mouse tissue inhibitor of metalloproteinases-3 (TIMP-3) gene and have identified a putative p53 binding site (5'-GGGCTTGCTT GACGTCCA GAACAGGGTC-3'), which contains two p53 consensus binding motifs (bold) with two nucleotide mismatches (underlined) in the second motif and an 8 bp spacer in between. Since both p53 and TIMP-3 are involved in cell cycle progression, we tested the hypothesis that TIMP-3 is a p53 downstream effector gene, mediating p53 activity. A good correlation between p53 protein levels and TIMP-3 expression was found among mouse liver cell lines. However, when TIMP-3 promoter driven luciferase constructs were tested for p53 responsiveness in these cells, the construct containing the putative p53 binding site did not show significant difference from the one having the p53 site deleted. The gel retardation assay showed that the oligo (T3) made from the putative p53 binding site in the mouse TIMP-3 promoter did not bind to p53 protein, nor did an oligo (T3W) with a correction for the two mismatched nucleotides in the second motif. When the 8 bp spacer was removed, however, the oligo T3WSF (same as the T3W with spacer free) but not T3SF (T3 without spacer) binds to p53, indicating that both the spacer between two motifs and consensus binding sites determined the p53 binding. It is worth noting that under a less stringent assay condition (0.2 microg instead of 1.0 microg of dI/dC), T3SF did weakly bind to p53. Lastly, the compounds that induce p53 transactivation activity did not induce TIMP-3 expression. We concluded from this study that TIMP-3 is not a p53 downstream effector gene.