Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanisms by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G 2 /M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G 1 arrest in addition to arresting cells at G 2 /M. Here we describe the mechanism of the apigenin-induced G 1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 µM apigenin resulted in dose-dependent cell-cycle arrest at both the G 0 /G 1 and G 2 /M phases as measured by flow cytometry. The G 0 /G 1 arrest was more clearly defined by using HDF that were synchronized in G 0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 µM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 µM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G 1 phase rather than in a G 0 quiescent state. The G 1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenintreated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 µM apigenintreated cells. In conclusion, apigenin treatment produced a G 1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.