Tumor necrosis factor α (TNFα) has been shown to be a potent stimulator of prostaglandin (PG) F_2α secretion in the bovine endometrium. The aims of the present study were to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGF_2α in response to TNFα, and the intracellular mechanisms of TNFα action. Cultured bovine epithelial and stromal cells were exposed to TNFα (0.006–6 nM) or oxytocin (100 nM) for 4 h. TNFα resulted in a dose-dependent increase of PGF_2α production in the stromal cells (P < 0.001) but not in the epithelial cells. On the other hand, oxytocin stimulated PGF_2α output in the epithelial cells but not in the stromal cells. When the stromal cells were incubated for 24 h with TNFα and inhibitors of phospholipase (PL) C or PLA₂, only PLA₂ inhibitor completely stopped the actions of TNFα (P < 0.001). When the stromal cells were exposed to TNFα and arachidonic acid, the action of TNFα was augmented (P < 0.001). When the stromal cells were incubated for 24 h with a nitric oxide (NO) donor (S-NAP), S-NAP stimulated the PGF_2α production dose-dependently. Although an NO synthase (NOS) inhibitor (l-NAME) reduced TNFα-stimulated PGF_2α production, an inhibitor of phosphodiesterase augmented the actions of TNFα and S-NAP (P < 0.05). The overall results indicate that the target of TNFα for stimulation of PGF_2α production in cattle is the endometrial stromal cells, and that the actions of TNFα are mediated via the activation of PLA₂ and arachidonic acid conversion. Moreover, TNFα may exert a stimulatory effect on PGF_2α production via the induction of NOS and the subsequent NO-cGMP formation.