Common killifish, or mummichogs (Fundulus heteroclitus), are a species of estuarine teleost that are widely used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions, widespread distribution, and relatively sedentary nature, makes them ideal as sentinel species of estuarine health. However, the lack of cell lines derived from F. heteroclitus places limitations on the utility of this species in environmental research. In contrast, cell cultures derived from other model organisms have assisted and facilitated our understanding of the effects that environmental contaminants have on organisms in vitro. The development and use of novel F. heteroclitus cell lines for toxicological and parasitological applications is reported here. Continuous proliferating cells were derived from pre-hatch embryos of killifish and have been maintained for 3 years. Three stable cell lines were obtained from the head and body tissues of F. heteroclitus; these stable cell lines have been dubbed KilliFish Embryo 1, 3, and 5 (KFE-1, KFE-3, KFE-5). All three cell lines have been characterized for origin and functionality, as well as for applications in toxicology, studying effects of model chemical pollutants, and in parasitology to evaluate a cod-infecting microsporidia that has been an emerging pathogen of concern, for their ability to infect and grow in cell lines derived from sentinel species. KFE-1 has characteristics of neuroepithelial cells, whereas KFE-3 are possibly liver derived cells, and KFE-5 are distinctly myogenic, as this line has cells that appear to be striated muscle cells. Like intact F. heteroclitus, these cell lines can withstand a wide temperature range from 4°C to 37°C. Mechanisms of thermotolerance and ability to withstand salinity and hypoxia as well as chemical toxicity tolerance could be readily studied with these new cell lines.