We studied the metabolic rate, cellular energetic state, hypoxia-inducible factor-1 (HIF-1) activation and expression of enzymes involved in energy metabolism, using rainbow trout (Oncorhynchus mykiss) hepatocytes over the oxygen range from 21 to 1 kPa. Oxygen-dependence of these factors was assessed by gradually reducing oxygen supply to cells from 21 kPa to 10, 5, 2 and 1 kPa. Moreover, time course experiments for up to 20 hours at oxygen tensions of 1 and 2 kPa were carried out. Reduction of oxygen from 21 kPa to 10, 5, 2 and 1 kPa decreased metabolic rate of the cells by 14, 24, 37, and 46 %, respectively. This response was instantaneous and fully reversible upon reoxygenation. Cellular ATP content and the expression of all mRNAs studied decreased when oxygen was reduced from 21 to 5 and 2 kPa. The lowest ATP levels, approximately 43 % of the initial value, were measured at 5 kPa of oxygen whereas the reduction in mRNA amounts was most pronounced at 2 kPa. At 1 kPa oxygen tension, both ATP content and mRNA amounts returned to normoxic (21 kPa) levels with a concomitant activation of HIF-1, indicating reorganization of energy metabolism in adaptation of cells to low oxygen supply. These results show that oxygen has a direct regulatory effect on metabolism of trout hepatocyte cultures supporting the view that oxygen has a profound role in metabolic regulation in cells.