Vitellogenin (Vtg) is an important precursor yolk protein in egg-laying vertebrates, including fish. The 17-oestradiol (E2) plays a crucial role in the Vtg synthesis; moreover, certain hormones can stimulate Vtg synthesis. We investigated the possible role of E2, carp recombinant growth hormone (crGH), insulin (Ins), progesterone (P4) and 11-deoxycortisol (11-DOC) hormones in Vtg synthesis on primary juvenile Chalcalburnus tarichi hepatocyte culture. The amount of Vtg in the medium was measured at 2-day intervals using enzyme-linked immunosorbent assay (ELISA). The hepatocytes were maintained in culture for more than 2weeks without the addition of serum components. Vitellogenin localization was visualized with the immunofluorescence method in E2-supplemented hepatocytes. Among hormones applied to the culture, only E2 had an influence on Vtg synthesis in a time-dependent manner, while crGH, Ins, P4 and 11-DOC had no effect. However, in hepatocytes stimulated with E2 in combination with P4, a lower Vtg production was seen compared with Vtg produced when hepatocytes were stimulated with E2 alone. P4 proved to have potentiating effects on co-treatment with E2-induced Vtg production. As a result, E2 and P4 are the most important hormones for Vtg synthesis in juvenile C. tarichi hepatocyte culture.