In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6- 2 H 5 ]-testosterone and [16,16,17- 2 H 3 ]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [ 2 H ]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [ 2 H 5 ]-Testosterone was converted into [ 2 H 4 ]-oestradiol-17β, [ 2 H 4 ]-oestrone and [ 2 H 3 ]-6-dehydro-oestradiol-17α in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse. On the basis of GC–MS characteristics ( M + m/ z 477/ 482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17α was recorded in dynamic incubations (twin peaks in the mass spectrum at m/ z 504/ 507, the molecular ion M +). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [ 2 H 4 ]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed. In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [ 2 H 3 ]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/ z 504/ 506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/ z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.