Characteristics of the transport of the nitric oxide synthase substrate l‐arginine and its inhibitor, N^G‐nitro‐l‐arginine (l‐NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l‐arginine and l‐NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l‐NOARG (650 pmol/mg of protein) was three to four times higher than that of l‐arginine (170 pmol/mg of protein). l‐NOARG uptake showed biphasic kinetics (K_{m 1} = 0.72 mM, V_{max 1} = 0.98 nmol/min/mg of protein; K_{m 2} = 2.57 mM, V_{max 2} = 16.25 nmol/min/mg of protein). l‐Arginine uptake was monophasic with a K_m of 106 µM and a V_max of 0.33 nmol/min/mg of protein. l‐NOARG uptake was selectively inhibited by l‐NOARG, N^G‐nitro‐l‐arginine methyl ester, and branched‐chain and aromatic amino acids. l‐Alanine and l‐serine also inhibited l‐NOARG uptake but with less potency. Uptake of l‐arginine was selectively inhibited by N^G‐monomethyl‐l‐arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l‐NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l‐arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l‐NOARG and l‐arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.