Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODCSMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in a-actin and myosin, maker proteins of differentiation, growth of ODCSMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of a-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of a-actin and myosin, resulting in differentiation through inhibition of focal adhesions. © Springer-Verlag 2011.