p53 is a transcriptional activator and repressor, but recent evidence suggests that some of its many biological functions may not be dependent on transcription. To determine whether p53 exerts a direct influence on nuclear DNA replication, purified human p53 was added to a transcription-free DNA replication extract from Xenopus eggs. Full-length human p53 that inhibits SV40 DNA replication in vitro had no effect on nuclear DNA synthesis in the Xenopus system. In contrast, a C-terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration-dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei. This inhibition occurred primarily at initiation of DNA synthesis. Oxidation of p53 delta 30, which eliminates DNA binding activity, also abrogated the protein's ability to inhibit nuclear DNA synthesis. The p53 binding DNA consensus sequence enhanced rather than competed away inhibitory activity of p53 delta 30. Therefore, p53 that is constitutively active for DNA binding can inhibit nuclear DNA replication in the absence of transcription. This inhibition may require binding of p53 to DNA, in addition to interactions between p53 and proteins of the replication complex.