Incubation of hepatocytes with D-galactosamine (GalN) produced a dose-dependent alteration in cell viability and a fall in ATP and fructose 2,6-bisphosphate (Fru-2,6-P2) levels. The reduction in Fru-2,6-P2 can be explained by changes in the substrates or modulators of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the adenosine 3',5'-cyclic monophosphate level nor the activity ratio of the enzyme was modified. Microcalorimetric measurements showed that GalN produced an exothermic peak followed by a progressive decrease in heat dissipation. Simultaneous administration of GalN and fructose 1,6-bisphosphate (Fru-1,6-P2) significantly increased cell viability, and concentrations of ATP and Fru-2,6-P2 and led to stable heat production. In the presence of Fru-1,6-P2 alone, hepatocytes kept ATP and Fru-2,6-P2 levels constant, whereas they increased the oxygen uptake-to-heat output ratio. Our results suggest that GalN initiates the hepatotoxic effect by means of an energy-dissipating interaction, produced before its metabolism and presumably at the membrane level, whereas Fru-1,6-P2 protects the cells against this injury in a way that prevents the initial interaction and increases the metabolic efficiency of the cell.