The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T₄), and 3,5,3′-triiodothyronine (T₃) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T₄ on VTG uptake were observed. Incubation of ovarian fragments with VTG and T₃ (T₃ group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T₃ group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (~150 μm), whereas T₃ showed these effects only in larger sized oocytes (>180 μm). These results suggest that both SPG and T₃ can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.