Cell cycle progression in mammalian cells is regulated by a family of cyclin-dependent kinases (cdks) that are activated by a family of 3 cdc25 phosphatases: cdc25A, cdc25B and cdc25C. We examined the expression of mRNA and protein of all 3 cdc25s during the HeLa cell cycle, and found that cdc25B protein has a unique and limited pattern of expression relative to other cdc25 homologs. Antisense oligonucleotides reduced cdc25B mRNA levels and dysregulated protein expression, while inhibiting S-phase progression in synchronized HeLa cells. Scrambled control oligonucleotides had no effect. Antisense oligonucleotides transfected in early G2-phase had no effect on cell cycle progression. A direct correlation between down-regulation of cdc25B and inhibition of thymidine incorporation was found using several oligonucleotides. Our results suggest a role for cdc25B in S-phase and demonstrate that inhibition of cdc25B has persistent antiproliferative effects.