Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMΦ) and tracheal epithelial cells, and whether l-arginine–dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [³H]-l-arginine, nitrite accumulation, and the turnover of [³H]-l-arginine by nitric oxide (NO) synthase and arginase (formation of [³H]-l-citrulline and [³H]-l-ornithine, respectively) were studied. Poly-l-arginine reduced [³H]-l-arginine uptake in rat AMΦ and tracheal epithelial cells in a concentration-dependent manner (at 300 μg/ml by 70%). Poly-l-lysine, protamine, and major basic protein (each up to 300 μg/ml) tested in rat AMΦ inhibited [³H]-l-arginine uptake by 35 to 50%. During 6 h incubation in amino acid–free Krebs solution, rat AMΦ, precultured in the absence or presence of LPS (1 μg/ml), accumulated 1.4 and 3.5 nmol/10⁶ cells nitrite, respectively. Addition of 100 μM l-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMΦ, respectively. Nitrite accumulation in the presence of l-arginine was reduced by poly-l-arginine and poly-l-lysine (100 and 300 μg/ml) by 60 to 85% and 20 to 30%, respectively. Poly-l-arginine, but not poly-l-lysine, inhibited nitrite accumulation already in the absence of extracellular l-arginine. Poly-l-arginine (300 μg/ml) inhibited [³H]-l-citrulline formation by AMΦ stronger than that of [³H]-l-ornithine. We conclude that cationic proteins can inhibit cellular transport of l-arginine and this can limit NO synthesis. Poly-l-arginine inhibits l-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.