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Establishment of cell lines from different tissues of different teleost fishes has remarkably increased. Development of fish cell lines has significantly contributed to recent advances in many research areas. Poecilia reticulata also known as guppy is a teleost fish which is widely used for ecotoxicological research. In this study to isolate fibroblast cell from caudal tissue of P. reticulata and to develop a cell line. For this purpose caudal tissue samples were collected from ten male P. reticulata under aseptic conditions. Primary culture was performed with tissue explant technique. Primary cells fed with L15 culture medium supplemented with FBS. To investigate the effects of different incubation temperature on proliferation rate of cells, seven different incubation temperatures (15°C, 20°C, 25°C, 28°C, 30°C, 32°C, 37°C) were assessed. Same experimental design was used for the effects of different FBS concentration (5%, 10%, 15%, and 20%) on proliferation rate of cells. Optimum growth rate was observed at 28°C temperature and 10% FBS concentration. Cells were subcultured succesfuly more than 60 times and this resulted in development of a cell line named as PSF. Cultured cells were also succesfuly cryopreserved. The revival rate of PSF cell line was up to %85-90 and this result could be admitted as a good when compared to other fish cell lines.