An investigation was made of hydroxy-steroid dehydrogenase activity in homogenates of rabbit ovaries from which the corpora lutea, if present, had been removed. Activity was determined by measuring the increase in optical density with time due to the formation of reduced pyridine nucleotide in incubation media saturated with oxidized pyridine nucleotide and hydroxysteroid. Activity was found toward steroids with hydroxyl groups in the 3[beta], 17[beta], 20 [alpha] and 20[beta] configurations. Both 3[beta]- and 17[beta]-hydroxysteroid dehydrogenase activities were stable for several days at either 3-5 or -20 C and were shown to increase from pH 8.6 to 10.0. The 3[beta]-hydroxysteroid dehydrogenase utilized DPN+ preferentially and was localized in the microsomal fraction, whereas the 17[beta]-, 20[alpha]- and 20[beta]-hydroxysteroid dehydrogenases preferred TPN+ and were found in the soluble fraction of an ovarian homogenate. The 3[beta] -hydroxysteroid dehydrogenase activity (m[mu]/mole/min/mg wet tissue) was found to be directly related to the weight of the ovary minus its corpora lutea, and so presumably to the amount of interstitial tissue present. Relative activities found with various steroids are reported.