The yeast Cdc14 phosphatase has been shown to play an important role in cell cycle regulation by dephosphorylating proteins phosphorylated by the cyclin-dependent kinase Cdc28/clb. We recently cloned two human orthologs of the yeast CDC14, termed hCDC14A and -B, the gene products of which share approximately 80% amino acid sequence identity within their N termini and phosphatase domains. Here we report that the hCdc14A and hCdc14B proteins interact with the tumor suppressor protein p53 both in vitro and in vivo. This interaction is dependent on the N termini of the hCdc14 proteins and the C terminus of p53. Furthermore, the hCdc14 phosphatases were found to dephosphorylate p53 specifically at the p34(Cdc2)/clb phosphorylation site (p53-phosphor-Ser(315)). Our findings that hCdc14 is a cyclin-dependent kinase substrate phosphatase suggest that it may play a role in cell cycle control in human cells. Furthermore, the identification of p53 as a substrate for hCdc14 indicates that hCdc14 may regulate the function of p53.