The effects of progesterone, its agonists (progestin RU-5020, glucocorticoid RU-26988) and antagonist (antiprogesterone, anti-glucocorticoid RU-486) were tested on isolated fat cells in vitro. When added to the incubation medium, all four steroids decreased basal glucose oxidation. The inhibitory effect of the steroids appeared early (20 min incubation) and was sustained during a 2-h incubation. The early inhibitory effect was less marked for progesterone agonist RU-5020 than for the other three steroids. When incubation was prolonged for 2 h, the lowest inhibitory effect was observed with progesterone antagonist RU-486. Insulin-stimulated glucose oxidation was inhibited by progesterone, its antagonist RU-486, one of its agonists RU-26988, but not by the other agonist RU-5020. Analysis of the dose response curves showed that progesterone, RU-26988, and RU-486 decreased fat cells' responsiveness and, only for RU-486, sensitivity to insulin. Adipocytes isolated from ovariectomized, progesterone-treated rats showed a decreased maximal response to insulin and decreased insulin sensitivity in opposition to cells incubated directly with the steroid. No inhibition of 125I-labeled insulin binding was seen as an acute or chronic effect of progesterone. It is concluded that progesterone and the studied related steroids decrease glucose oxidation by mechanism(s) distal to insulin binding to its specific receptors.