Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F-1; mature) and third-largest (F-3; developing) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. The amounts of basal bronectin and progesterone produced by granulosa cells from F-1, F-3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F-1 and F-3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F-1 and F-3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F-1 and F-3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.