Aims: To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene. Methods and Results: A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real-time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP-dependent GDH activity. NADP-GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP-GDH activity under temperature, pH and NaCl values found during cheese ripening. RT-PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down-expressed by low temperature (< 13 degrees C) and over-expressed by NaCl (1 center dot 87-5 center dot 62%). According to NADP-GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49. Conclusions: The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese-related bacteria. Significance and Impact of the Study: It focuses on the phenotypic and molecular characterization of the NADP-GDH in lactobacilli under cheese-ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.