The antiproliferative activity of alkylated heparin, in which the terminal end of heparin is derivatized with an alkyl group (butyl, octyl, lauryl, stearyl), was examined using vascular smooth muscle cells. The proliferation of cells, which were growth-arrested prior to addition of heparin, was inhibited in proportion to both increase in the chain length of the alkyl group of alkylated heparin and alkylated heparin concentration in the serum-containing medium. The antiproliferative activity of stearyl group derivatized heparin was significantly stronger than that of nonmodified heparin. Little proliferation was observed at high dose (500 mug/mL). Confocal laser scanning microscopic observation indicated that alkylated heparin was accumulated on the cell membranes at an early incubation time, followed by homogeneous distribution of intracellular space. The therapeutic potential of alkylated heparin for preventing restenosis after balloon angioplasty is discussed.