The endothelial nitric oxide synthase (NOS3) gene has been implicated in the pathogenesis of hypertension-related left ventricular hypertrophy (LVH). Candidate-gene studies have examined the role of NOS3 variation, but reported results are inconsistent. In this study, we investigated the association of three clinically relevant polymorphisms (promoter T786C, intronic 4a/b, and nonsynonymous G894T) in a case-control sample of 230 ethnically homogeneous (Caucasians) patients with essential hypertension, with (n = 64) and without (n = 166) clinically diagnosed LVH. Haplotype analysis was also performed. In single-marker analyses, no significant associations with LVH were detected by univariate and multivariate regression models. In the haplotype-based association analysis, no common haplotype was associated with the development of LVH. A rare haplotype consisting of the three mutant alleles (C-a-T*) was found to be present only in patients with LVH (3.4%) and not in control hypertensive patients. Despite the biological rationale for the involvement of the NOS3 gene in LVH, no evidence for a major role of common NOS3 haplotypic variation was found. Considering the totality of available evidence, single-gene analyses of the NOS3 gene have not uncovered detectable genetic effects, and pathway-based analyses that examine interactions of multiple loci may be more informative about the complex genetic etiology of LVH.