The in vitro culture of fully grown mammalian oocytes results in spontaneous meiotic maturation from prophase arrest to metaphase II. This maturation can be inhibited by steroid hormones in both murine and porcine oocytes. Using selected steroids, we have examined the structure-activity relationships of steroids and oocyte inhibition. Experiments with androgens, estrogens, glucocorticoids, and progesterone revealed that at least one steroid from each class was inhibitory. Progesterone, however, was two to three times more effective than steroids from other classes. Examination of a variety of progestins showed that most substitutions decreased or abolished the inhibitory activity. Hydroxy group substitutions at different carbon atoms and substitutions at the 4-ene group lessened the inhibitory effectiveness, with the exception of 5 beta-dihydroprogesterone, which was as effective as progesterone. However, several steroids with substitutions at the C17 acetyl group were more active than progesterone, including 20 beta-dihydroprogesterone which was the most inhibitory steroid tested (ID50 = 5 microM). The progesterone agonist R5020 was also very active (ID50 = 8 microM). This is the first report of a detailed examination of the steroid-induced inhibition of murine oocytes. A comparison between the results reported here and previous reports of steroid-induced inhibition in porcine oocytes reveals differences in the response of oocytes from the two families. The structure-activity relationships of the inhibitory steroids examined here suggest that the steroids are acting via a receptor-mediated system.