The tumour suppressor p53 has been shown to regulate RNA polymerase (pol) III transcription both in vitro and in vivo. We have characterized the regions of p53 that contribute to this effect. Repression of pol III transcription in vivo does not require residues 13-19 near the N-terminus of p53 that are highly conserved through evolution. However, amino acids 22 and 23 in the adjacent transactivation domain do contribute to the inhibition of pol III activity. Deletions within the central DNA-binding core domain (residues 102-292) of p53 can entirely abolish the repression function in these assays, despite the fact that pol III templates contain no recognized p53 binding site. Deletion or substitution within the C-terminal domain of p53 can also compromise its ability to repress pol III activity in vitro and in transfected cells. These observations reveal that repression of pol III transcription is a complex function involving multiple regions of p53 extending throughout much of the protein.