Measurement of steroids in saliva has excited interest because of the numerous potential clinical applications; noninvasive, convenient sampling; and apparently accurate reflection of the concentrations of physiologically active unbound steroid in the circulation. Although assays of saliva for several steroid hormones are available and widely used, assays for salivary estradiol are not, primarily because of methodological limitations. By modifying a commercially available kit for serum estradiol, our laboratory has developed a procedure that is sensitive, highly specific, and reliable for measuring salivary estradiol. Assay sensitivity is 0.5 fmol (0.14 pg; sample concentration 1.3 pmol/L) with a mean interassay CV of 10.8% at low concentrations. Clinical studies showed that values for serum and saliva are highly correlated (P less than 0.001), and demonstrated reliable detection of estradiol peaks during normal ovulatory cycles in serial samples from 15 women. Salivary estradiol peaked at 5.4 (SD 1.9) pmol/L on cycle day 14.4 (SD 3.2), 1.2 (SD 0.8) days before ovulation detected by ultrasound. This assay may be particularly helpful in investigating ovarian function and free estradiol in women at various stages of the reproductive cycle.