Using antibody-coated test tubes as a detection method, three types of assay for 17β-estradiol (E2) have been investigated. Method A involved the measurement of E2 in the dried ether extract of biological fluids. Method B required a chromatographic step after ether extract, prior to measurement of E2. Method C (direct method) measured E2 directly in the biological fluid, diluted with assay buffer. Method C was found reliable for the measurement of E2 in human follicular fluid, but not in pregnancy serum. Method A was adequate for pregnancy serum, but overestimated E2 in serum from normal adult male and female subjects. Method B was reliable to measure E2 in all the biological fluids tested.