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Transforming Growth Factor-beta (TGFβ) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFβ has any effect on G2 checkpoint kinases. We have found that TGFβ downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24 h for cyclin B1 and cdc2 and by 48 h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFβ which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2–pRb complexes, which was decreased to 30% of control levels by 48 h after initiating TGFβ treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFβ. In addition, TGFβ caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2–3 h before a dramatic decrease of both molecules by 48 h. Taken together, our data suggest that TGFβ has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFβ-induced G1 arrest and growth inhibition.