Isolated stromal cells from the ampullary and isthmic parts of bovine oviductal tissues were cultured in monolayer and spheroid (cell aggregate) systems. Prostaglandin F2 alpha (PGF) plays a crucial role in oviductal contraction and is produced by oviductal epithelial cells in cattle. Since stromal cells of many organs produce PGF, PGF production by bovine oviductal stromal cells was investigated. After PGF synthesis was confirmed, the utility of isolation and culture methods for oviductal stromal cells was evaluated by PGF production in the present study. The homogeneity of the cells was > 99%. PGF production of the cells was increased by tumor necrosis factor-a. The stromal cells aggregated and farmed a spheroid by the treatments with several reagents. PGF production was higher in the spheroid culture than in the monolayer culture. The isolation and culture methods described here will facilitate studies of the physiological function of bovine oviductal stromal cells.