The present study was designed to establish the cellular localization and expression of transforming growth factor β (TGFβ) signaling pathway components, including TGFβ1 and β2; TGFβ receptors type I (TβRI) and II (TβRII); and Smads 2, 3, 4, and 6 during gonadotropin-induced follicular maturation and ovulation in the mouse ovary. Immature 21-day-old mice were sequentially treated with recombinant human FSH, 5 IU daily for 3 days, and hCG once at Day 24 of life. Immunohistochemical experiments revealed a TGFβ1 staining in granulosa cells (GC) and theca interna cells (TIC) as well as in oocytes, whereas that of TGFβ2 was mainly localized in oocytes and GC. Strong immunostaining for both TβRI and -RII was observed in the TIC and, to a lesser extent, in GC. Whereas oocytes did not exhibit any staining for TβRII, their TβRI immunostaining was strong. Smads were detected in oocytes, GC, and luteal cells and in a lesser amount in TIC; the immunostaining for Smad 4 was the strongest. Western blotting and reverse transcription-polymerase chain reaction analyses indicated that, in response to gonadotropins, TGFβ2, TβRI, Smad 2 and Smad 4 mRNA and protein levels increased, while those of Smad 6 decreased in ovarian homogenates. In conclusion, these results show that, in a model of immature mouse exposed to a sequential gonadotropin treatment, FSH and LH increased the expression of the TGFβ signaling system through the increase of TGFβ2, TβRI, stimulatory Smad 2, and common Smad 4 expression, which occurred concomitantly with a decrease of the inhibitory Smad 6 expression.