Ectonucleotide pyrophosphatase/phosphodiesterase 3 ( Enpp3) is involved in multiple physiological processes, such as morphological changes and inflammatory processes. The present study investigated the spatiotemporal expression pattern and regulatory mechanisms controlling expression of Enpp3 in the rat ovary during the periovulatory period. Immature female rats were injected with pregnant mare serum gonadotropin to stimulate follicular development. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after human chorionic gonadotropin (hCG) administration. Real-time polymerase chain reaction analysis revealed that messenger RNA (mRNA) for Enpp3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. In situ hybridization analysis demonstrated that Enpp3 mRNA expression was induced in theca cells at 4 hours after hCG, and the expression remained elevated until 12 hours after hCG. The expression of Enpp3 mRNA was stimulated in granulosa cells at 8 hours and reached the highest expression at 12 hours. Localization of Enpp3 mRNA was observed in newly forming corpora lutea by in situ hybridization. The hCG-stimulated expression of Enpp3 mRNA was blocked by a protein kinase C inhibitor (GF109203) instead of the protein kinase A inhibitor (H89). Furthermore, Enpp3 induction is dependent on new protein synthesis. Inhibition of progesterone action did not alter Enpp3 mRNA expression, whereas inhibition of prostaglandin synthesis or the epidermal growth factor pathway diminished Enpp3 mRNA levels. In conclusion, our findings suggest that the induction of the Enpp3 mRNA may be important for the morphological changes and inflammatory response during ovulation and luteinization.