Sam68 (Src-associated in mitosis 68 kDa) is a protein that associates with and is tyrosine phosphorylated by Src in a mitosis-specific manner, thereby raising the possibility of a role for Src in the regulation of the cell cycle. This study examines the effects of radicicol, a Src tyrosine kinase inhibitor, upon both the phosphorylation of Sam68 and mitotic progression in Src-transformed mouse fibroblasts. Radicicol reversibly inhibits the mitosis-specific tyrosine phosphorylation of Sam68 in vivo, as determined by antiphosphotyrosine immunoblotting. Radicicol inhibits the tyrosine phosphorylation of both free and Src-associated Sam68, suggesting the presence of two intracellular pools of tyrosine phosphorylated Sam68 in mitotic cells. In addition, radicicol treatment has no effect on the ability of cells to enter mitosis, indicating that tyrosine phosphorylation of Sam68 is probably not important for cells to enter mitosis. However, radicicol reversibly retards the exit of cells from mitosis, as determined by flow cytometric analyses. Radicicol mediated inhibition of Sam68 tyrosine phosphorylation, and its concurrent ability to block mitotic exit suggests the possibility of a significant role for Src kinase and this unique mitotic substrate, Sam68, in cell cycle regulation.