Previous studies have demonstrated a protective effect of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 against atherosclerosis and restenosis, two disorders characterized by abundant proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts. These therapeutic effects might result from p27Kip1-dependent suppression of both cell proliferation and migration. However, the interplay between cell growth and locomotion remains obscure. We show here that p27Kip1 inhibits cellular changes that normally occur during cell locomotion (eg, lamellipodia formation and reorganization of actin filaments and focal adhesions). Importantly, a p27Kip1 mutant lacking CDK inhibitory activity failed to inhibit vascular smooth muscle cell and fibroblast proliferation and migration. Moreover, a constitutively active mutant of the retinoblastoma protein (pRb) insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration. In contrast, inactivation of pRb by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Collectively, these results suggest that cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/CDK/pRb pathway. These findings might have important implications for the development of novel therapeutic strategies targeting the fibroproliferative/migratory component of vascular occlusive disorders.