Glutamine synthetase (GS) converts ammonia and glutamate into glutamine. We assessed the activity of the 5′ regulatory region of the GS ene in developing transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of 3150 bp of the upstream sequence of the rat GS gene to obtain insight into the spatiotemporal regulation of its pattern of expression. To determine the organ-specific activity of the 5′ regulatory region CAT and GS mRNA expression were compared by ribo-nuclease-protection and semi-quantitative in situ hybridization analyses. Three patterns were observed: the 5′ region is active and involved in the regulation of GS expression throughout development (peri-central hepatocytes, intestines and epididymis); the 5′ region shows no activity at any of the ages investigated (periportal hepatocytes and white adipose tissue); and the activity of the 5′ region becomes repressed during development (stomach, muscle, brown adipose tissue, kidney, lung and testis). In the second group, an additional element must be responsible for the activation of GS expression. The last group included organs in which the 5′ regulatory region is active, but not in the cells that express GS. In these organs, the activity of the 5′ regulatory region must be repressed by other regulatory regions of the GS gene that are missing from the transgenic onstruct. These findings indicate that in addition to the 5′ regulatory region, at least two unidentified elements are involved in the spatioteniporal pattern of expression of GS.