Nitric oxide (NO), produced by endothelial cells, is a signaling molecule synthesized from l-arginine by nitric oxide synthases (NOS). NO is known to reduce the ratio of receptor activator of nuclear factor KappaB (RANKL)/osteoprotegerin (OPG), leading to decreased osteoclastogenesis and a reduction in bone resorption. Endothelial nitric oxide synthase (eNOS or NOS3) is the predominant constitutive isoform of nitric NOS within bone. Recently, a NOS3 polymorphism, Glu298Asp, previously implicated in osteoporosis, failed to demonstrate an association with bone mineral density (BMD), although there was some indication of an association with selected geometry indices. Since a single polymorphism does not capture all of the potential variants in a given gene, we investigated a broader coverage of the NOS3 gene with bone density/ultrasound and geometry indices in a sample of unrelated individuals from the Framingham Offspring Study. Our results indicated that the Glu298Asp polymorphism was not associated with BMD but suggested some haplotype-based associations in the linkage disequilibrium (LD) region that included the Glu298Asp polymorphism with several geometry indices. Although our findings exhibited several associations with selected bone density/ultrasound and geometry indices, the nominally significant associations are regarded as primarily hypothesis generating and suggest that replication in other samples is needed. Thus, NOS3 genetic variation does not appear to be a major contributor to adult bone density/ultrasound and geometry in our sample.