Gas6 (encoded by growth-arrest-specific gene 6) is a γ-carboxyglutamic acid (Gla)-containing protein which is released from growth-arrested vascular smooth muscle cells (VSMCs) and potentiates VSMC proliferation induced by Ca²⁺-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importance of Gla residues for the biological activities of Gas6 and tried to assess the importance of endogenous Gas6 in VSMC proliferation. We demonstrated that Gla-deficient Gas6 lacked receptor-binding and growth-potentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next we used warfarin, an inhibitor of vitamin K-dependent γ-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of the vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC proliferation but not that induced by epidermal growth factor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca²⁺-mobilizing growth factors.