Objective This study investigated the impact of stress on effectors of the l-arginine/nitric oxide (NO) system including the endogenous inhibitor asymmetric dimethylarginine (ADMA). Methods Black (n = 168) and white (n = 206) South African teachers were exposed to a mental and a physical stressor for 1 minute, respectively. Serum samples for determination of l-arginine, NO metabolites, ADMA, and symmetric dimethylarginine (SDMA) were obtained at rest and during stress exposure. Perception of task stressfulness was assessed on a 7-point Likert scale, and psychological distress was estimated by the General Health Questionnaire. Results Black South Africans exhibited higher resting levels of NO metabolites (adjusted mean [standard error of the mean] = 11.3 [1.3] versus 3.9 [1.1] μmol/l, p < .001) but lower circulating ADMA (0.62 [0.02] versus 0.70 [0.02] μmol/l, p = .004) and SDMA (0.41 [0.01] versus 0.53 [0.01] μmol/l, p < .001) than did white South Africans. Ethnicity-by-psychological distress interaction was observed for resting levels of ADMA (p = .002), SDMA (p = .038), and l-arginine (p = .048). Ethnic differences in responses to experimental stress were evident for NO metabolites (blacks versus whites: 5.94 [1.55] versus -0.74 [1.25] μmol/l, p = .004) and SDMA (blacks versus whites: -0.02 [0.01] versus 0.02 [0.01] μmol/l, p = .004). Ethnicity-by-psychological distress interaction for stress responses was found for l-arginine/ADMA ratio (p = .027). Conclusions The l-arginine/NO system is affected by psychosocial distress with higher susceptibility in black South Africans. This interaction may contribute to the higher cardiovascular disease risk in black South Africans.