Background and Purpose In the German Multicenter Erythropoietin (EPO) Stroke Trial, patients not receiving thrombolysis most likely benefited from EPO on clinical recovery, whereas a combination of rtPA and EPO was associated with increased mortality. We investigated whether the combination of rtPA and EPO increased release of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA), and thereby potentially deteriorated ischemic stroke outcome, as suggested from experimental data. Methods ADMA was determined in serum samples from 90 patients of the German Multicenter EPO Stroke Trial taken at days 1 (within 6 hours after symptom onset), 2, 3, 4, and 7 after stroke using high-performance liquid chromatography-tandem mass spectrometry. ADMA was analyzed for the different treatment groups (EPO, n=25; placebo, n=30; rtPA+placebo, n=18; EPO+rtPA, n=17). Clinical outcome was expressed as difference between National Institutes of Health Stroke Scale at baseline and 90 days. Results ADMA levels significantly increased during the observation time in EPO, EPO+rtPA, and placebo groups (P<0.05). A treatment effect on ADMA levels was revealed by repeated measures ANOVA only in the rtPA+placebo group (P=0.027). Here, ADMA levels were decreased compared with the placebo group (P<0.05). Both the EPO and the rtPA+placebo groups in the Hannover subgroup of the EPO trial had better outcome than the placebo group (P<0.05). Conclusions Our data underscore the potential benefit of EPO in ischemic stroke. The hypothesis from experimental data, that EPO treatment increases ADMA in stroke patients, was disproved. Further studies are needed to clarify whether decreased ADMA might contribute to therapeutic rtPA effects.