Our previous work has shown associations between childhood adiposity and perinatal methylation status of several genes in umbilical cord tissue, including endothelial nitric oxide synthase (eNOS). There is increasing evidence that eNOS is important in bone metabolism; we therefore related the methylation status of the eNOS gene promoter in stored umbilical cord to childhood bone size and density in a group of 9-year-old children. We used Sequenom MassARRAY to assess the methylation status of two CpGs in the eNOS promoter, identified from our previous study, in stored umbilical cords of 66 children who formed part of a Southampton birth cohort and who had measurements of bone size and density at age 9 years (Lunar DPXL DXA instrument). Percentage methylation varied greatly between subjects. For one of the two CpGs, eNOS chr7:150315553 + , after taking account of age and sex, there were strong positive associations between methylation status and the child's whole-body bone area (r = 0.28, P = 0.02), bone mineral content (r = 0.34, P = 0.005), and areal bone mineral density (r = 0.34, P = 0.005) at age 9 years. These associations were independent of previously documented maternal determinants of offspring bone mass. Our findings suggest an association between methylation status at birth of a specific CpG within the eNOS promoter and bone mineral content in childhood. This supports a role for eNOS in bone growth and metabolism and implies that its contribution may at least in part occur during early skeletal development.