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Background: The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development. Methods: Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2-18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1-2 mL), early (testis, 3-6 mL), mid (testis, 8-12 mL), late-1 (testis,15-25 mL, not reached final height) and late-2 (testis,15-25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L. Results: Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5-95 percentiles: <4 - 7) pmol/L) to early puberty (6 (<4 - 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 - 17) pmol/L) and late-1 (21 (12 - 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 - 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h. Conclusion: With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys. [ABSTRACT FROM AUTHOR]