Agmatine is a biogenic amine with the capacity to regulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian cell lines and herein characterize agmatine transport in mammalian cells. In transformed NIH/3T3 cells, agmatine uptake is energy dependent with a saturable component indicative of carrier-mediated transport. Transport displays an apparent Michaelis-Menten constant of 2.5 µM and a maximal velocity of 280 pmol · min¹ · mg¹ protein and requires a membrane potential across the plasma membrane for uptake. Competition with polyamines, but not cationic molecules that utilize the y⁺ system transporter, suppresses agmatine uptake. Altering polyamine transporter activity results in parallel changes in polyamine and agmatine uptake. Furthermore, agmatine uptake is abrogated in a polyamine transport-deficient human carcinoma cell line. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. The fact that this transport system is associated with proliferation could be of consequence to the antiproliferative effects of agmatine.