Abstract Background Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N -acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. Results Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N -acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli Δ argA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035 , which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli Δ argA mutant in vivo and homologous overexpression led to a significantly higher intracellular N -acetylglutamate pool. Enzyme assays confirmed the N -acetylglutamate synthase activity of Cg3035 in vitro . However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. Conclusions The N -acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum . It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae , comprising amongst others the genera Corynebacterium , Mycobacterium , and Nocardia . Therefore, the name C-NAGS ( Corynebacterineae -type NAGS) is proposed for this new family.