Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P₄), androstenedione (A₄), estradiol-17β (E ₂) and Prostaglandin F_2α (PGF _2α) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P₄ and PGF_{2 α} were similar among the different sizes of follicles. The release of A₄ was observed in middle and large follicles. E₂ concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A₄ release. PGF_2α release was considerably high after LH infusion compared to the control group. Infusion of PGF_2α increased P₄ and A ₄ release but there was no change in E₂ release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF_2α seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.