Effect of extracellular calcium in vitellogenin (VTG) production in response to estradiol-17 $eta$ $(E_2,;2 imes10^{-6}M)$ was examined in primary hepatocyte culture of rainbow trout, Onchorhynchus mykiss. Total calcium in estrogenized sera significantly increased, compared with the control, while diffusible calcium was insignificant. However, diffusible calcium in the incubation medium with $E_2$ was significantly reduced, compared with the control. The uptake of extracellular calcium by cultured hepatocytes signifIcantly increased 90 min after $E_2$ addition. Moreover, the accumulation of intracellular calcium increased in the cultures with $E_2$, regardless of the calcium concentrations in the incubation media. In addition, $E_2-primed $ VTG production was significantly decreased by withdrawal of E_2$ from the incubation medium. Moreover, VTG production by $E_2-primed$ hepatocytes was reduced by removing calcium from the incubation medium with or without $E_2$. These results suggest that the entry of extracellular calcium into the cytoplasm is an important step for VTG production in primary hepatocyte cultures in rainbow trout.