The yeast URA2 locus encodes a multifunctional protein which possesses the carbamylphosphate synthetase and aspartate transcarbamylase activities and which catalyzes the first two reactions of the pyrimidine pathway. We report here the nucleotide sequence of the central and the 3' region of this locus. The latter encodes that part of the multifunctional protein which has the aspartate transcarbamylase activity. The deduced amino acid sequence shows a high degree of homology with the known aspartate transcarbamylases of various organisms from Escherichia coli to mammals. The amino acid residues that have been shown to be involved in the catalytic site of the E. coli enzyme are all conserved suggesting that, in the more complex structure of the yeast protein, the catalytic sites are also located at subunit interfaces. There is also an important conservation of the amino acid pairs that, in E. coli, are implicated in intra- and interchain interactions. As well as the oligomeric structure suggested by these two features, the three-dimensional structure of the yeast enzyme must also be organized to account for the channeling of carbamylphosphate, from the carbamylphosphate synthetase catalytic site to that of aspartate transcarbamylase, and for the concomitant feedback inhibition of the two activities by the end product UTP. The URA2 gene product was shown to be localized in the nucleus. With the aim of identifying the regions that may be involved in this transport, we have determined by electron microscopy the subcellular distribution of aspartate transcarbamylase in three strains expressing different fragments of the URA2 locus. In the first strain the protein lacks 190 residues at the N terminus, but accumulates normally in the nucleus. In the second strain the protein lacks 382 residues in the central part and seems impaired in the nuclear transport process. In the third strain the 476-residue protein encoded by the 3' region of URA2 locus and catalyzing the aspartate transcarbamylase reaction is able by itself to migrate to and accumulate in the nucleus. This suggests that two regions are involved in the nuclear accumulation. On the basis of their conservation in analogous proteins of other eukaryotes and their similarity to sequences already identified as nuclear location signals, a sequence in the central region of the protein and two short sequences in the C-terminal region are good candidates for the nuclear location signal involved in the targeting of the URA2 product.