While L-arginine is clearly recognised as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y⁺ transport is established, little data exists regarding the acute regulation of this transport process. We specifically investigated the effect of acetylcholine (ACh) and isoprenaline (ISO) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady state intra-arterial (i.a.) infusion of [³H]L-arginine (100nCi.min^-1), the effects of ACh (9.25 and 37µg.min^-1), ISO (25-50 and 200µg.min^-1) and sodium nitroprusside (SNP) (1-2 and 8µg.min^-1) on forearm plasma flow (FPF), [³H]L-arginine uptake and [³H]L-citrulline release were determined. In parallel experiments, the effects of ACh, ISO and SNP on [³H]L-arginine uptake were studied in BAEC. L-arginine uptake was inversely related to FPF (r=-0.50; p<0.005). At a similar FPF (ACh 56.82±9.25, ISO 58.49±5.56, SNP 57.92±4.96 ml.min^-1; p=ns), i.a. ACh significantly increased forearm uptake of [³H]L-arginine (54655±8018dpm.min^-1), compared to that observed with either ISO (40517.23±6841dpm.min^-1; p=0.01) or SNP (36816±4650dpm.min^-1; p=0.011). This was associated with increased ACh-induced [³H]L-citrulline release, when compared to ISO and SNP (p=0.046). Similarly, in BAEC, ACh significantly increased [³H]L-arginine uptake, compared to control, ISO or SNP (ACh 12.0x10⁷±1.83x10⁷ vs. Control 6.67x10⁷±1.16x10⁷ vs. ISO 7.35x10⁷±1.63x10⁷ vs. SNP 6.01x10⁷±1.11x10⁷ fmol.min^-1.mg^-1 at 300µmol.L^-1 L-arginine; p=0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.