Liver is one of the most important organs in vertebrates that have important roles in detoxifying. This organ was used as a target organ in many physiological and toxicological aspects. The main purpose of the present study was developing appropriate methodology for the primary cultivation of hepatic cells from orange-spotted Grouper, Epinephelus coioides, a subtropical fish species of the family Serranidae. In present study, hepatocytes were isolated from five grouper individuals. Initially, the fish wiped with 70% ethanol. Liver were removed and cut into small pieces with scissors and hepatocytes were disconnected using different enzymatic digestion with collagenase (Type 1 and 4) and trypsin and additional nutrient materials in culturing mediums. Then, cells were cultured for 2 weeks in Lebowitz L-15 under 3 methods: 1. using enzymatic digestion by trypsin, 2. using enzymatic digestion by collagenase (type 1 and 4) and 3. Using nutrients and additives was cultured. Finally, effects of different incubation temperature (20, 25, 28, 30 and 32 degree of Celsius) and Bovine serum content (0, 10 and 20% and 20%+ITS) on cell growth were estimated. According to the results, digestion by collagenase type 4, resulted in more cell colonization and growth in comparison with other methods. At the same method, cells showed fibroblastic morphology. In conclusion, the best culture method for primary hepatocyte from orange-spotted Grouper, Epinephelus coioides, was using ITS+20%FBS under 30 degree of Celsius incubation temperature
