Ovarian cancers are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation is associated with ovulation and has been suggested to play a role in ovarian surface transformation and cancer progression. Aspects of ovarian surface repair after ovulation include proliferation, migration, and surface regeneration. To study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 d of culture. Organ cultures were propagated in medium supplemented with BSA and fetal bovine serum to determine optimal growth conditions. BSA cultured organs had OSE that proliferated significantly more than controls until d 4, whereas fetal bovine serum cultured organs had significantly more surface area encapsulated by OSE. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer.