Progesterone plays an essential role in the maintenance of the pregnancy of most mammals. 20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD) catalyses the inactivation of progesterone into its inactive form, 20alpha-hydroxyprogesterone, and could thus be involved in progesterone withdrawal and in the control of gestation. In this report, the purification and crystallization of recombinant human and rabbit 20alpha-HSDs (h20alpha-HSD and rb20alpha-HSD) are described, two highly homologous enzymes possessing, in addition to their common 20alpha-HSD activity, different activities and substrate specificities. Complete diffraction data sets have been collected for crystals of rb20alpha-HSD in complex with NADP(H) and with either dihydrotestosterone (1.8 A), progesterone (1.7 A) or 4-androstenedione (1.8 A). All these crystals belong to the monoclinic space group P2(1). A partial data set has also been collected for a crystal of h20alpha-HSD (P2(1)2(1)2(1)) in complex with NADP(H) and progesterone.