In Saccharomyces cerevisiae, the ARG5/ARG6 locus encodes both acetylglutamate kinase and acetylglutamyl-phosphate reductase, localized in the mitochondria. Genetic analysis, determination of the nucleotide sequence of the ARG5/ARG6 gene and identification of the transcript indicate that it encodes a single translation product containing two enzyme activities. However, analysis of cellular extracts revealed that the activities are completely separable. In this work, we define different domains in the ARG5/ARG6 polypeptide; a mitochondrial target sequence and the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains. We show that deletions in the N-terminal end of the protein and point mutations in the junction region between the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains lead to the accumulation of large precursor. Our data support the idea that import of the ARG5/ARG6 precursor into the mitochondria is required for its processing into two mature enzymes.