Age-related changes affecting mitochondrial adenine nucleotide metabolism may underlie age-related decreases in hepatic metabolic activities. Oxidative activity coupled with phosphorylation, the apparent Km and Vmax of the adenine nucleotide translocase (AdNT), the adenine nucleotide pool size and membrane lipid composition were determined for hepatic mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. The age-related decreases in state 3 respiration supported by NAD-linked substrates were 2-4-fold greater than that of an FAD-linked substrate. The 32% (P less than 0.05) decrease in the AdNT Vmax calculated for the aged rats was accompanied by a 17% decrease in the AdNT Km. The exchangeable pool of adenine nucleotides in mitochondria from aged rats was 72% (P less than 0.05) that in the young rats. While the age-related increase in the cholesterol/phospholipid-Pi ratio and changes in the phospholipid head group pattern were not significant, the overall change in the fatty acid pattern effected a 20% (P less than 0.05) decrease in the n-6/n-3 fatty acid ratio. These data suggest that the reduced Vmax of the AdNT is a consequence of a diminished pool of exchangeable adenine nucleotides. The lower AdNT velocity may reflect the effect of changes in the lipid environment of the membrane in which it is embedded. The major shifts in these parameters occurred during the second year of life.