A primary epithelial cell line, DK1, established from renal tissue of a spontaneously aborted female Atlantic bottlenose dolphin was transfected with linearized pSV3.neo, an SV40 virus-derived plasmid encoding large tumor antigen (Tag). Transfected cells were grown in cetacean culture medium supplemented with 400 microg/ml geneticin (G418), and individual clones were selected using cloning rings. DKN1 was the first clone to be evaluated for future research use, and has been continuously cultured for 8 years. Intracellular cytokeratin and the expression of Tag were determined in DKN1, and cell growth was evaluated under different concentrations of l-glutamine, glutathione, and N-acetylcysteine. DKN1 cells did not require high levels of l-glutamine as previously reported for cetacean cells, and addition of antioxidants at the concentrations used in this study (2.0mM) decreased the rate of cell division. These data suggest strongly that these immortalized bottlenose dolphin epithelial cells have different levels of, and requirements for, glutathione than would be considered normal for terrestrial mammalian cells, do not require high levels of l-glutamine as previously suggested for dolphin cells, and exhibit decreased levels of cell growth and viability in high levels of the antioxidant GSH and its precursor, NAC.