This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor mRNA, play specific roles during follicular development. In particular, we analyzed the presence of the ED-I region, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN whereas total FN levels remained relatively constant. A negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels. This steroid was without effect on the alternative splicing of FN in primary cultures of bovine granulosa cells. However, cAMP produced a marked decrease in the incorporation of the ED-I region. In contrast, transforming growth factor beta (TGF-beta) elicited both a stimulation on overall FN synthesis and an increase in the inclusion of ED-I. This effect was evident at the protein level (Western blots) and also in the mRNAs (Northern blots). A peptide corresponding to the ED-I region stimulated DNA synthesis in a bovine granulosa cell line (BGC-1) whereas the peptide corresponding to the flanking sequences was without effect. Data presented herein suggest a novel form of regulation by which changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.