The present study was designed to establish the cellular localization and expression of transforming growth factor beta (TGFbeta) signaling pathway components, including TGFbeta1 and beta2; TGFbeta receptors type I (TbetaRI) and II (TbetaRII); 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 TGFbeta1 staining in granulosa cells (GC) and theca interna cells (TIC) as well as in oocytes, whereas that of TGFbeta2 was mainly localized in oocytes and GC. Strong immunostaining for both TbetaRI and -RII was observed in the TIC and, to a lesser extent, in GC. Whereas oocytes did not exhibit any staining for TbetaRII, their TbetaRI 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, TGFbeta2, TbetaRI, 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 TGFbeta signaling system through the increase of TGFbeta2, TbetaRI, stimulatory Smad 2, and common Smad 4 expression, which occurred concomitantly with a decrease of the inhibitory Smad 6 expression.