Because fibronectin is a major secretion product of rat granulosa cells in culture, we have investigated 1) the involvement of endogenous fibronectin in granulosa cell attachment, and 2) the consequences of inhibition of this attachment on follicle-stimulating hormone (FSH)-dependent differentiated responses. Attachment was significantly inhibited for up to 8 h in a concentration-dependent manner by antiserum to rat fibronectin, but not by nonimmune serum. Adsorption of antiserum on fibronectin or addition of exogenous fibronectin eliminated this inhibitory activity. Treatment with antiserum did not significantly alter the FSH-dependent production of adenosine 3',5'-cyclic monophosphate over 2 h or progestins over 48 h, while conversion of testosterone to 17 beta-estradiol over 48 h was suppressed by 60% in the presence of antiserum, regardless of antiserum adsorption on fibronectin. Results indicate that endogenous fibronectin is involved in substratum attachment of rat granulosa cells, but that attachment is not a requisite for FSH responsiveness.