Background and ObjectivesBoth genetic and environmental factors contribute to stroke risk. We sought to identify novel metabolites associated with incident stroke in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort and determine whether they reflected genetic or environmental variation.MethodsThis was a stroke case–cohort observational study nested in REGARDS. Cases were defined as incident stroke and metabolomic profiles were compared to a randomly selected control cohort. In baseline plasma samples, 162 metabolites were measured using liquid chromatography–tandem mass spectrometry. Cox proportional hazards models were adjusted for age, sex, race, and age by race in the base model. Fully adjusted models included traditional stroke risk factors. Mediation analyses conducted for these stroke risk factors used the metabolite as mediator. Genome-wide associations with the leading candidate metabolites were calculated using array data. Replication analyses in the Jackson Heart Study (JHS) were conducted using random effects meta-analysis.ResultsThere were 2,043 participants who were followed over an average period of 7.1 years, including 1,075 stroke cases and 968 random controls. Nine metabolites were associated with stroke in the base model, 8 of which were measured and remained significant in meta-analysis with JHS. In the fully adjusted model in REGARDS, guanosine (hazard ratio [HR] 1.34, 95% CI 1.18–1.53; p = 7.26 × 10−6) and pseudouridine (HR 1.28, 95% CI 1.13–1.45; p = 1.03 × 10−4) were associated with incident ischemic stroke following Bonferroni adjustment. Guanosine also partially mediated the relationship between hypertension and stroke (17.6%) and pseudouridine did not mediate any risk factor. Genome-wide association analysis identified loci rs34631560 and rs34631560 associated with pseudouridine, but these did not explain the association of pseudouridine with stroke.DiscussionGuanosine and pseudouridine are nucleosides associated with incident ischemic stroke independently of other risk factors. Genetic and mediation analyses suggest that environmental exposures rather than genetic variation link nucleoside levels to stroke risk.Classification of EvidenceThis study provides Class II evidence that guanosine and pseudouridine are associated with incident stroke.