Nitric oxide synthase 3 (NOS3) catalyzes production of NO in the endothelium and may play a role in cardiovascular disease (CVD). We assessed the pharmacogenetic associations of three NOS3 polymorphisms and three antihypertensive drugs with CVD outcomes. Hypertensive subjects (n = 30,280) from a multi-center, double-blind clinical trial were randomized to chlorthalidone, amlodipine, or lisinopril treatment (mean follow up, 4.9 years). Outcomes included coronary heart disease (CHD: fatal CHD and nonfatal myocardial infarction); stroke; heart failure (fatal, requiring hospitalization, or outpatient treatment); all-cause mortality; and end-stage renal disease (ESRD). Main effects of NOS3 variants on outcome and genotype-treatment interactions were tested. For NOS3 -690 C>T (rs3918226), a higher hazard ratio (HR) was found in minor allele carriers for CHD (CC = 1.00, CT+TT = 1.12 (95% confidence interval (CI) = 1.00-1.26), P = 0.048). For NOS3 -922 A>G (rs1800779), a higher HR was found in minor allele carriers for heart failure (AA = 1.00, AG+GG = 1.10 (CI = 1.00-1.21), P = 0.046). Significant pharmacogenetic findings were observed for stroke and all-cause mortality. For -690 C>T, a lower HR was observed for stroke in minor allele carriers when treated with amlodipine versus lisinopril (CC = 0.85 (CI = 0.73-0.99), CT+TT = 0.49 (CI = 0.31-0.80), P = 0.04). For glu298asp G>T (rs1799983), a lower HR was observed for all-cause mortality in minor allele carriers when treated with amlodipine versus lisinopril (GG = 1.01 (CI = 0.91-1.13), GT+TT = 0.85 (CI = 0.75-0.97), P = 0.04). We observed significant associations with NOS3 variants and CHD and heart failure and significant pharmacogenetic effects for stroke and all cause mortality. This suggests that NOS3 variants may potentially provide useful clinical information with respect to treatment decisions in the future.