The role of progesterone in luteinizing hormone- (LH) induced fibronectin production and deposition by chicken ovarian granulosa cells was examined in vitro. Granulosa cells isolated from pre-ovulatory follicles of the domestic hen ovary were incubated in serum-free Medium 199, and the total amount of fibronectin (deposited, secreted into the medium and associated with cells) produced was measured by ELISA. LH increased the amount of fibronectin deposited by granulosa cells. Similarly, it increased the quantity of fibronectin secreted into the medium or associated with cells. Cyanoketone (an inhibitor of progesterone synthesis) suppressed dose dependently basal and LH-induced fibronectin deposition. Cyanketone also attenuated the total amount of fibronectin produced by control or LH-stimulated granulosa cells. Exogenous progesterone reversed the inhibitory effects of cyanoketone on basal and LH-induced fibronectin production and deposition. The non-degradable synthetic progestin R5020 stimulated fibronectin production in a dose-dependent manner. R5020 also reversed the inhibitory effects of cyanoketone on LH-induced fibronectin production and deposition. The antiprogestin, ZK 98.299, inhibited basal and LH-stimulated fibronectin production. The data demonstrate that endogenous progesterone regulates fibronectin production and deposition perhaps in an intracrine/autocrine manner. They indicate that LH-stimulated fibronectin production and deposition by chicken granulosa cells is mediated (at least in part) by progesterone.