Nitric oxide (NO) is synthesized from L-arginine (Arg) by NO synthase (NOS), and asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. Normal distribution values of Arg, ADMA, and SDMA are required to evaluate the effects of cardiovascular drugs on blood vessels, but insufficient normal reference values from rat and mouse plasma exist for new drug development and screening. To determine the means and variations in the basal endogenous materials concentration, Arg, ADMA, and SDMA in blank rat (n = 24) and mouse (n = 37) plasma samples were quantified using LC-MS/MS equipped with an electrospray ionization interface to generate positive mode ions. Accuracy and precision were within 90.42-110.91%, and 0.88-13.84%, respectively, for analyses of Arg, ADMA, and SDMA. The average plasma concentrations of Arg, ADMA, and SDMA were 175.38 +/- 13.87 microM, 0.79 +/- 0.20 microM, and 0.84 +/- 0.20 microM, respectively, in rats and 70.81 +/- 19.38 microM, 0.66 +/- 0.21 microM, and 0.42 +/- 0.10 microM, respectively, in mice. These results will provide a basis on which to evaluate cardiovascular drug effects on ARG, ADMA, and SDMA levels in new drug development.