The cdc25+ gene of fission yeast encodes a phosphotyrosine phosphatase that dephosphorylates tyrosine-15 of p34cdc2 and thereby activates p34cdc2/cyclin to bring about entry into M phase. We have recently cloned a human homolog, CDC25, which rescues the M-phase initiation defect of yeast cdc25 temperature-sensitive mutants. Antibodies raised against the CDC25 gene product specifically recognize human proteins of approximately 55 and approximately 52 kDa. Microinjection of affinity-purified anti-CDC25 antibodies into HeLa cells inhibits entry into mitosis. These observations suggest that the CDC25 gene products are essential for the initiation of mitosis in human cells, similar to their homologs in fission yeast and Drosophila. CDC25 gene products, like p34CDC2, are localized primarily in the nucleus during interphase, suggesting that activation of p34CDC2/cyclin by p52/p55CDC25 occurs within the nucleus.