The development of an ovarian follicle requires a complex set of reciprocal interactions between the oocyte and granulosa cells in order for both types of cells to develop properly. These interactions are largely orchestrated by the oocyte via paracrine factors such as growth differentiation factor 9 (GDF9). To examine these interactions further, a study was conducted of the effects of oocytes at different stages of development on proteins synthesized by mouse granulosa cells during the transition of granulosa cells (GCs) from preantral, secondary (2°) follicles (2° GCs) to mural granulosa cells (3° GCs) of antral tertiary (3°) follicles. The ability of recombinant GDF9 to mimic the effects of oocytes was also determined. Effects were evaluated by high- resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis. Coculture of the 2° GCs with growing oocytes (GOs) from 2° follicles brought about many of the changes in granulosa cell phenotype associated with the 2° to 3° follicle transition. GDF9 likewise brought about many of these changes, but only a subset of GDF9-affected protein spots were also affected by coculture with GOs. Coculture of 2° GCs with the nearly fully grown oocytes (FGOs) from 3° follicles had a reduced effect on 2° GC phenotype, in comparison with coculture with GOs. For some proteins, oocyte coculture or GDF9 treatment appeared to have opposite effects on 2° GCs and 3° GCs. Additional effects of GDF9 and oocytes were seen in cultures of 2° GCs for proteins other than those that differed between untreated control 2° and 3° GCs. These results indicate that GOs and GDF9 can each induce 2° GCs to shift their phenotype toward that of 3° GCs. The ability of the oocyte to produce this effect is diminished with oocyte development. The transition in the GC phenotype promoted by oocytes appears stable because differences in 2° GCs promoted by oocytes and GDF9 were observed in untreated 3° GCs. We conclude that the influence of the oocyte on GCs changes with the progression of their development, and so too does the response of the GCs to the oocyte. Moreover, by acting on the 2° GCs, GOs are able to influence stably the phenotype of 3° GCs. Thus, at or near the 2° to 3° follicle transition, signals from the growing oocyte contribute to the development of the mural GC phenotype.