Single intact corpora lutea were isolated from pseudopregnant rat ovaries stimulated with PMSG and HCG. The activity of 20α-hydroxvsteroid-dehydrogenase (20α-OH-SDH) was measured at various stages of pseudopregnancy. The influence of PGF 2α or a metabolically stable PGF 2α-derivative on this enzyme in vitro was assessed. 20α-OH-SDH activity was low from day 6 to 8, started to rise on day 10 and reached a maximum on day 14 after administration of HCG. The enzyme could be stimulated by the PGF 2α -derivative on day 10 and 11 but not on day 6, 8, when basal enzyme activity was still very low or on 12 and 14 of pseudopregnancy when the enzyme had already reached maximal levels. PGF2 or its derivative had no effect on enzyme activity in α cytosolic fraction indicating that particulate cell constituents were required for this process. PGE 2 as well as cycloheximide (an inhibitor of protein biosynthesis) were without influence on enzyme stimulation. dB-cAMP but not dB-cGMP decreased enzyme activity. Ca 2+-ions were required for the stimulation of 20α-OH-SDH activity. It is concluded that PGF2 or its stable derivative may modulate ovarian steroid Metabolism by acute stimulation of luteal 20α-OH-SDH.