The ansamycin antibiotic herbimycin A is a potent tyrosine kinase inhibitor and reduces the growth rate of various types of mammalian cells. When quiescent Rat6 fibroblast cells were treated with herbimycin A, serum-induced expression of cyclin D1 was inhibited, and this was associated with inhibition of G1 phase progression. However, herbimycin A also inhibited serum-induced G1 progression in derivatives of the Rat6 fibroblast cell line that stably overexpress a human cyclin D1 cDNA (RGccnD1#4 cells), without affecting the expression levels of G1 cyclins. We found that herbimycin A prevented serum-induced downregulation of the cyclin-dependent kinase inhibitor p27(kip1) thereby leading to inactivation of the protein kinase activity of CDK2. These results suggest that herbimycin A inhibits a tyrosine kinase(s) that plays a role in degradation of the p27(kop1) protein.