Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5mm) and small follicles (SF, 3-5mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.