L-arginine transport is crucial for macrophage activation as it supplies substrate for the key enzymes nitric oxide synthase 2 and arginase I. These enzymes participate in classical and alternative activation of macrophages, respectively. Classical activation of macrophages is induced by type I cytokines and alternative activation is induced by type II cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF), in addition to inducing proliferation and differentiation of macrophages activates arginase I, but its action on L-arginine transport is unknown. Here we studied the L-arginine transporters that are active in mouse primary bone marrow-derived macrophages (BMM) and examined the effect of GM-CSF treatment on transport activities. Under basal conditions, L-arginine entered mainly through system y⁺L (>75%). The remaining transport was explained by system y⁺ (<10%) and a diffusion component (10-15%). In response to GM-CSF treatment, transport activity increased mostly through system y⁺ (over 10 fold), accounting for about 40% of the total L-arginine transport. The increase in y⁺ activity correlated with a rise in CAT2 mRNA and protein. Furthermore, GM-CSF induced an increase in arginase activity and in the conversion of L-arginine to ornithine, citrulline, glutamate, proline and polyamines. BMM obtained from CAT2 knock-out mice responded to GM-CSF by increasing arginase activity and the expression of CAT1 mRNA, which also encodes system y⁺ activity. Nonetheless, the increase in CAT1 activity only partially compensated the lack of CAT2 and L-arginine metabolism was hardly stimulated. We conclude that BMM present mainly y⁺L activity and that, in response to GM-CSF, L-arginine transport augments through CAT2, thereby increasing the availability of this amino acid to the cell.