High-protein diets exacerbate glomerular hyperfiltration and the progression of diabetic nephropathy. The purpose of this study was to determine whether amino acids also produce nonhemodynamic injury in the glomerulus. When rat mesangial cells were cultured with an amino acid mixture designed to replicate the composition in plasma after protein feeding, production of mRNA (Northern blot analysis) and/or protein (ELISA or Western blot analysis) for transforming growth factor-β1 (TGF-β1), fibronectin, thrombospondin-1 (TSP-1), and collagen IV were enhanced in a manner comparable to a culture with high glucose (30.5 mM). The bioactive portion of total TGF-β (NRK assay) increased in response to amino acids. The TSP-1 antagonist LSKL peptide reduced bioactive TGF-β1 and fibronectin, indicating the dependence of TGF-β1 activation on TSP-1. DNA synthesis ([3H]thymidine incorporation), an index of cellular proliferation, increased in response to amino acids and was further enhanced by culture with increased levels of both amino acids and glucose. TGF-β1 and matrix proteins increased when mesangial cells were cultured with excess L-arginine (2.08 mM) alone. Although L-arginine is the precursor of nitric oxide (NO), such responses to amino acids do not appear to be mediated through increased NO production. NO metabolites decreased in the media, and these responses to mixed amino acids or L-arginine were not prevented by NO synthase inhibition. In conclusion, amino acids induce indicators of response to injury in mesangial cells, even when hemodynamic stress is absent. In conditions associated with increased circulating amino acids, such as diabetes and/or a high-protein diet, direct cellular effects could contribute to glomerular injury.