Glutamate dehydrogenase is an enzyme catalysing a reaction for ammonium assimilation, alternative to those performed by glutamine synthetase and glutamate synthase. In the genus Prochlorococcus, genomic studies have shown the presence of the gdhA gene (encoding glutamate dehydrogenase) in only four of the sequenced strains, including MIT9313. We studied the physiological regulation of glutamate dehydrogenase in this strain, by measuring the expression of gdhA, the intracellular concentration of the enzyme and its activity. Our goal was to clarify the physiological role of glutamate dehydrogenase, in order to understand why it has been selectively conserved in certain strains. Studies performed in cultures under nitrogen starvation, or with inhibitors of the nitrogen assimilation, suggest that the main role of glutamate dehydrogenase is not the assimilation of ammonium. Glutamate dehydrogenase activity and gdhA expression increased along the growth of cultures. Besides, we found a significant upregulation in gene expression when cultures were grown on glutamate as nitrogen source. We suggest that the main physiological role of glutamate dehydrogenase in Prochlorococcus MIT9313 is the utilization of glutamate to produce ammonium and 2-oxoglutarate, and amino acid recycling, thus enabling to use amino acids as nitrogen source. Therefore we propose that glutamate dehydrogenase is present in the genome of strains for whom the utilization of amino acids is most important.