Abstract Context Members of the TGF-β family have been implicated in aberrant follicle development in women with polycystic ovaries (PCO). Objective Are there quantitative differences in the concentrations of TGF-β family members in fluid from human small antral follicles (hSAFs) in women with or without PCO? Design and Setting Follicle fluids (FFs) were collected from 4- to 11-mm hSAFs obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Patients FFs from 16 women with PCO (FF = 93) and 33 women without PCO (FF = 92). Main Outcome Measures Intrafollicular concentrations of growth differentiation factor-9 (GDF9); anti-Müllerian hormone (AMH); inhibin-A and inhibin-B; total inhibin; activin-A, activin-B, and activin-AB; follistatin; follistatin-like-3; estradiol; and testosterone. Results Activin-B concentrations were reported in hSAFs, and concentrations were 10 times higher than activin-A and activin-AB concentrations. Activin-B showed significant associations with other growth factors. Concentrations of inhibin-A and inhibin-B were significantly lower in FFs from women with PCO, especially in hSAFs <8 mm in diameter. AMH concentrations did not differ between the groups in hSAFs <8 mm; however, AMH remained high in hSAFs >8 mm in women with PCO but decreased in women without PCO. Estradiol was significantly lower in FFs from women with PCO and showed significant associations with AMH. Concentrations of GDF9 showed significantly higher concentrations in PCO FFs of follicles >6 mm. Conclusions Altered concentrations of TGF-β family members in hSAFs from women with PCO highlight altered growth factor signaling as a potential mechanism for follicle growth arrest.