HT-29 Glc-/+ cells originate from a human colon adenocarcinoma. These cells have been selected in a glucose-free culture medium and switched back in a glucose-containing medium. In this condition, they can spontaneously differentiate after confluency in enterocyte-like cells according to the activity of the brush-border associated hydrolase dipeptidyl peptidase IV. Since L-arginine can generate polyamines which are necessary for cellular proliferation and also differentiation, and nitric oxide with reported anti-proliferative property, the metabolism of this amino acid was examined in proliferative and differentiated isolated HT-29 cells. Proliferative HT-29 cells were characterized by micromolar intracellular concentration of putrescine and millimolar concentration of spermidine and spermine. In these cells, L-arginine is converted to L-ornithine and putrescine and to a minor part to nitric oxide and L-citrulline. Putrescine was taken up by HT-29 cells, leading to the production of a modest amount of spermidine. The diamine was slightly incorporated into cellular proteins and largely released in the incubation medium. The proliferative HT-29 cells take up spermidine and spermine but do not catabolize these polyamines and slightly released spermidine. Differentiation of HT-29 cells is not associated with change in intracellular polyamine content but is paralleled by an almost complete extinction of de novo synthesis of putrescine (due to a dramatic decrease of ornithine decarboxylase activity) and by a reduced release capacity of putrescine. In contrast, putrescine net uptake and incorporation into cellular proteins remained unchanged after differentiation. Furthermore, spermidine and spermine metabolism as well as the circulation of L-arginine in the nitric oxide synthase pathway were also not modified after differentiation. In conclusion, putrescine is the L-arginine-derived molecule, the metabolism of which is specifically and markedly modified when HT-29 cells move from proliferative to differentiated state.