Under the protection of ascorbic acid a 4-hydroxyoestrone-bovine serum albumin conjugate was prepared containing intact 4-hydroxyoestrone as determined by gas chromatography-mass spectrometry. Using this antigen, antibodies with high affinity and specificity for 4-hydroxyoestrone were raised in rabbits. An assay procedure for the determination of 4-hydroxyoestrone in human urine and the assessment of its reliability are described. The following urinary excretion rates were found: male children 0.29 microgram/24 h, men (less than 50 microgram/24 h, men (20-40 years) 1.6 microgram/24 h, men (less than 50 years) 1.8 microgram/24 h, women, follic, 2.0 microgram/24 h, pre-ov. 5.3 microgram/24 h, luteal 2.4 microgram/24 h, women, pregnant, first trim. 30.0 microgram/24 h, second trim. 64.0 microgram/24 h, third trim. 48.0 microgram/24 h, women, post-men. 1.5 microgram/24 h. Thus the amounts of 4-hydroxyoestrone excreted in human urine are about 1/3 to 1/10 of those of 2-hydroxyoestrone. During the menstrual cycle the excretion rates of 4-hydroxyoestrone are in the same order of magnitude as those of oestradiol and show a clear-cut pre-ovulatory peak.