Several groups of investigators have shown that treatment of rats with glucagon produces an increase in the adenine nucleotide content of hepatic mitochondria. It has been suggested that this enlarged pool of exchangeable nucleotides may be responsible for several of glucagon's stimulatory effects on mitochondrial functions by accelerating the transport of adenine nucleotides across the inner mitochondrial membrane. This hypothesis was tested by loading rat liver mitochondria in vitro with adenine nucleotides to supranormal levels. This procedure did result in stimulation of several metabolic and bioenergetic functions including pyruvate carboxylation, uncoupler-dependent ATPase, and succinic dehydrogenase activity but not formation of citrulline. However, a sham loading that did not increase the nucleotide content of the mitochondria was essentially as effective as the loading procedure in stimulating those functions assayed. Mitochondria, loaded in vitro with supranormal levels of adenine nucleotides, were shown to have an enlarged pool of exchangeable nucleotides. This exchange was atractyloside sensitive, but the rate of exchange was only slightly increased as a consequence of enlargement of the pool. Similarly, mitochondria isolated from glucagon-treated rats showed no increase in the rate of exchange, although the exchangeable pool was increased. There was no correlation between the rate of nucleotide exchange and the rate of the uncoupler-dependent ATPase.