Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon arginine-starvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.