Objective Ultra-sensitive hormone assays have detected slight sex differences in blood estradiol (E₂) levels in young children before adrenarche. However, the origin of circulating E₂ in these individuals remains unknown. This study aimed to clarify how E₂ is produced in young girls before adrenarche. Design This is a satellite project of the Japan Environment and Children’s Study organized by the National Institute for Environmental Studies. Methods We collected blood samples from healthy 6-year-old Japanese children (79 boys and 71 girls). Hormone measurements and data analysis were performed in the National Institute for Environmental Studies and the Medical Support Center of the Japan Environment and Children’s Study, respectively. Results E₂ and follicle stimulating hormone (FSH) levels were significantly higher in girls than in boys, while dehydroepiandrosterone sulfate (DHEA-S) and testosterone levels were comparable between the two groups. Girls showed significantly higher E₂/testosterone ratios than boys. In children of both sexes, a correlation was observed between E₂ and testosterone levels and between testosterone and DHEA-S levels. Moreover, E₂ levels were correlated with FSH levels only in girls. Conclusions The results indicate that in 6-year-old girls, circulating E₂ is produced primarily in the ovary from adrenal steroids through FSH-induced aromatase upregulation. This study provides evidence that female-dominant E₂ production starts several months or years before adrenarche. The biological significance of E₂ biosynthesis in these young children needs to be clarified in future studies.