A novel fibroblast‐like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation of the stress activated member of the mitogen‐activated protein kinase family, p38MAPK and induction of heat shock protein (Hsp70) were evident. The time‐course of the p38MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38MAPK‐specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress.