Nitric oxide (NO) is synthesized from L-arginine (ARG) catalyzed by the enzyme nitric oxide synthase (NOS) and is important in the regulation of vascular tone, neurotransmission and host defense. N,N-Dimethyl L-arginine (asymmetric dimethylarginine, ADMA) and N-monomethyl L-arginine (MMA) are endogenous inhibitors of NOS. N,N'-Dimethyl L-arginine (symmetric dimethylarginine, SDMA), the inactive enantiomer of ADMA is also known to be present endogenously. A simple, sensitive and fast LC-MS-MS method was developed to extract and quantitate ADMA, SDMA, MMA and ARG from human plasma. 13C6-ARG was used as the internal standard for the assay. Protein precipitation using acetonitrile gave good recoveries of all the compounds from plasma. The compounds were separated by HPLC in less than 15 min using a silica column. The limits of detection for this method were found to be approximately 1 ng/ml for ARG, ADMA and SDMA and 2.5 ng/ml for MMA. The total LC-MS-MS analysis time is less than 15 min making this the fastest and most specific method reported to date. The use of an isocratic liquid chromatographic separation makes this method optimal for high sample throughput. The inter- and intra-day precision (% RSD) and accuracy (% error) for this assay were less than 15%. The average concentrations of ARG, ADMA, SDMA and MMA in plasma from 20 human subjects were found to be 10.9+/-4.1 microg/ml, 25.1+/-9.4 ng/ml, 33.2+/-13.1 ng/ml and 19.6+/-3.8 ng/ml, respectively.