Initial velocity measurements of [3H]ADP and [3H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent Vmax values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the Km and Vmax values for ATP uptake are greater than the Km and Vmax value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca2+, Mg2+, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca2+, long-chain acyl-CoA ester and AMP. Mitochondrial Ca2+ levels are elevated 3-5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28-38 and 6-16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4-5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca2+ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.