Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co-sediment with lipid raft/ caveolin-1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin-1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K-RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin-1 phosphorylation at tyrosine residue 14. Caveolin-1 is a negative regulator of caveolar endocytosis and phosphorylation in a K-RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K-RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin-dependent and clathrin-independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K-RAS in a caveolin-1 dependent manner.