The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 °C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes (cyp19a1, fshr, lhr and star). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle.