An analysis of the hormonal regulation of fibronectin production by granulosa cells showed that GnRH stimulated fibronectin secretion. GnRH increased fibronectin production 2.5- to 5-fold over that of control untreated cultures and greater than 20-fold over that of FSH-treated cultures. The GnRH concentration required for a minimal response was 10(-10) M, and that required for a maximal response was 10(-8) M. In contrast to the effects of GnRH, FSH suppressed fibronectin production to low or undetectable levels. In addition, FSH abolished the actions of GnRH on fibronectin production. Treatment of cultured granulosa cells with either (Bu)2cAMP or methylisobutylxanthine to influence cellular cAMP levels mimicked the actions of FSH on fibronectin secretion. Similar to FSH, both (Bu)2cAMP and methylisobutylxanthine abolished the actions of GnRH on fibronectin production. These results indicated that an increase in cAMP levels resulted in suppression of fibronectin secretion by granulosa cells and inhibition of the actions of GnRH. Insulin treatment of granulosa cells also suppressed fibronectin secretion, but to a lesser extent than FSH. In addition, insulin inhibited the actions of GnRH on fibronectin production by approximately 40-50% of stimulated levels. GnRH promoted a state of cytodifferentiation of the granulosa cell which had a high level of fibronectin production and, as shown previously, a low level of steroidogenesis. In contrast, FSH promoted a state of cytodifferentiation which had a low level of fibronectin production and a high level of steroidogenesis Both fibronectin and steroidogenic enzymes (e.g. aromatase) provide useful markers for an analysis of the cytodifferentiation of granulosa cells between these two distinct differentiated states. Results are discussed in relation to the possible mechanisms controlling granulosa cell cytodifferentiation and the possible functions of fibronectin in the ovary.