Regular exploitation of rubber tree causes intense metabolic activity in latex cells located in the phloem, associated with the regeneration of the latex cellular components removed by tapping. In this context, glutamine synthetase (GS) plays a prominent role by ensuring ammonium fixation necessary for the regeneration of latex nitrogenous components. In latex, GS activity was detected in the cytosol only. The enzyme was purified and its main physico-chemical characteristics were determined. Its molecular mass was 450 kDa for the native form and 45 kDa ± 5 kDa for the sub-unit. The pH optimum was found to be 8.0 and variations in the physiological pH range (6.4 to 7.2) strongly affected GS activity. Km for ammonium, glutamate and ATP were estimated at 8 microM ± 2 microM, 4 mM ± 0.5 mM and 0.6 mM ± 0.05 mM, respectively. Comparison with the cytosol physiological parameters showed the importance of ATP concentration for GS activity regulation. Functioning of the purified enzyme in a paraphysiological (ultrafiltrated, protein-free) medium, as compared with an optimised control medium, demonstrated that physiological ATP concentrations were significantly limiting for GS potential activity. Treatment with ethephon increases latex production both by increasing latex flow duration and by stimulating the regeneration of metabolism. In this paper we demonstrated that ethephon treatment induced a concomitant increase in pH, ATP concentration and GS activity. These results suggest that biochemical mechanisms involving pH and ATP participate in the regulation of GS activity in response to ethylene, in addition to the previously observed stimulation of gene expression.