The following evidence demonstrates that ammonia production in muscle occurs via the reactions of the purine nucleotide cycle: i) Extracts of cytosol which lack glutamine dehydrogenase produce ammonia under conditions that mimic muscle doing work. In such extracts a member of the purine nucleotide cycle (AMP, IMP, or adenylosuccinate) must be present in order that ammonia production can take place, ii) Perfused hindleg of rat or hindleg in situ produces ammonia during exercise, and there is a concomitant production of IMP and adenylosuccinate. Exercise causes a decrease in the contents of glutamate, aspartate, and glutamine and an increase in the content of alanine of perfused hindleg of rat. However, output of alanine does not change or is diminished during exercise. Glutamine output is diminished by exercise. Epinephrine increases the output and tissue content of ammonia and glutamine. It decreases the output and content of alanine, and it decreases the contents of aspartate and glutamate.