Somatic embryogenesis (SE) is a laboratory-based method that allows for cost-effective production of large numbers of clonal copies of plants, of particular interest for conifers where other clonal propagation methods are mostly unavailable. In this study, the effect of l-glutamine as an organic nitrogen source was evaluated for three contrasted media (containing NH4 + NO3 without glutamine, or glutamine + NO3, or glutamine without inorganic nitrogen) during proliferation and maturation of Norway spruce somatic embryos through analyses of activities of the key enzymes of nitrogen metabolism: nitrate reductase (NR), glutamine synthetase (GS) and arginase. A major change in nitrogen metabolism was indicated by the increased activity of GS from zero in the proliferation stage through maturation to high activity in somatic embryo-derived plantlets; furthermore, NR activity increased from zero at the proliferation stage to high activity in maturing embryos and somatic-embryo derived plantlets. In contrast, arginase activity was high at all stages. In accordance with the GS and NR data, proliferation was fastest on the glutamine media, the largest number of mature embryos developed on the glutamine medium without inorganic nitrogen, and the ammonium content of proliferating cultures was much higher than that of mature somatic embryos. The best developed embryos, judged by germination, were from the glutamine + NO3 medium. These insights into nitrogen utilization will help to improve culture medium composition.