To examine the mechanisms whereby amino acids modulate nitric oxide (NO) production and blood flow in the renal vasculature, chemiluminescence techniques were used to quantify NO in the renal venous effluent of the isolated perfused rat kidney as different amino acids were added to the perfusate. The addition of 10^-4 or 10^-3 M cationic amino acids (L-ornithine, L-lysine, or L-homoarginine) or neutral amino acids (L-glutamine, L-leucine, or L-serine) to the perfusate decreased NO and increased renal vascular resistance. Perfusion with anionic amino acids (L-glutamate or L-aspartate) had no effect on either parameter. The effects of the cationic and neutral amino acids were reversed with 10^-3 M L-arginine and prevented by de-endothelialization or NO synthase inhibition. The effects of the neutral amino acids but not the cationic amino acids were dependent on extracellular sodium. Cationic and neutral amino acids also decreased calcimycin-induced NO, as assessed by DAF-FM-T fluorescence, in cultured EA.hy926 endothelial cells. Inhibition of system y⁺ or y⁺L by siRNA for the cationic amino acid transporter 1 or the CD98/4F2 heavy chain diminished the NO-depleting effects of these amino acids. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space stimulate efflux of L-arginine out of the cell. Thus, the present experiments demonstrate that cationic and neutral amino acids can modulate NO production in endothelial cells by altering cellular L-arginine transport through y⁺ and y⁺L transport mechanisms.