The subcellular localization of glutamine synthetase in tobacco and the differential expression of two genes encoding cytosolic enzyme was investigated using both immunocytochemistry and in situ hybridization. Two full length cDNA clones each encoding cytosolic GS (Gln 1-3 and Gln 1-5) were isolated from a tobacco seeding cDNA library. A strong homology was found in the coding region of the two clones whereas the 3'- and 5'-untranslated sequences were dissimilar. In order to determine the levels of transcription, specific sequences from Gln1-3 and Gln1-5 were used in an RNAse protection assay. This experiment clearly showed that the gene encoding Gln1-3 is expressed in roots and flowers whereas the gene encoding Gln1-5 is transcribed at a high level in stems and at a lower level in roots and flowers. Immunogold labelling was used to examine the subcellular and cellular distribution of glutamine synthetase in vegetative and reproductive organs of tobacco plants. In mature leaf tissue or petals and sepals, plastidic GS was visualised only in the stroma matrix of chloroplasts and plastids. Cytosolic GS was detected in a number of vegetative or reproductive organs including leaves and flowers. In leaves cytosolic GS was preferentially located in the vascular tissue. In situ hybridization was performed using sections of tobacco organs and specific antisense RNA probes to the genes encoding Gln1-3 and Gln1-5. Gln1-5 transcripts were localised in the vascular tissues of stems and roots whereas Gln1-3 transcripts were detected in all root cells and floral organs including petals, sepals and anthers.