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1. Partially purified 17beta-hydroxy steroid-NAD(+) oxidoreductases, prepared from Pseudomonas testosteroni (EC 1.1.1.51), human term placenta (EC 1.1.1.62) and the cytoplasmic fraction of rat liver (EC 1.1.1.-) were tested for their ability to catalyse the oxidoreduction of 18-hydroxyoestradiol-17beta and 18-hydroxyoestrone. The products of incubation were identified by chromatographic procedures and by mass spectrometry. 2. The Pseudomonas enzyme catalysed both the oxidation of 18-hydroxyoestradiol-17beta and the reduction of 18-hydroxyoestrone; in contrast, the placental and rat liver enzymes only catalysed the reduction of 18-hydroxyoestrone. 3. These results were confirmed, by using a spectrophotometric assay; equimolar quantities of oestradiol-17beta and 18-hydroxyoestradiol-17beta were oxidized at approximately the same rate by the microbial enzyme. 4. These findings suggest that 18-hydroxyoestradiol-17beta may be a normal oestrogen metabolite. 5. The differences in ability of the mammalian and microbial enzymes to metabolize 18-hydroxylated oestrogens is explained on the basis of recognition sites with different geometrical dimensions, characteristic of the placental (Descomps & Crastes de Paulet, 1969) and the microbial steroid enzymes (Fosset & Crastes de Paulet, 1967).

1. In view of previous experiments in vitro and in vivo, in which 6-hydroxylation of phenolic steroids by various tissues was demonstrated, an attempt was made to isolate 6-hydroxy oestrogens from the urine of pregnant women. 2. During this work it was found that free 6alpha- as well as 6beta-hydroxylated oestrogens were extremely unstable under acidic conditions (pH <3); it was therefore necessary to establish experimental conditions under which no rearrangement of the hydroxyl group would take place. 3. By avoiding acid steps during the experimental procedures, a ketonic-phenolic fraction was obtained from enzymically hydrolysed late-pregnancy urine that was subjected to paper chromatography in various systems. 4. By using similar methods on a large scale and partition chromatography on Celite columns, a crystalline material was obtained from 1001. of late-pregnancy urine that was identified as 6alpha-hydroxyoestrone by various chemical reactions and by infrared spectroscopy.