Despite the growing interest in GTP-binding proteins and their role in higher plants, no compartimental analysis has been performed on tonoplast, until now. After successive extraction with a cushion of saccharose and a glycerol gradient, the tonoplast of Spinacia oleracea was checked by electron microscopy and the purity of the preparations verified by enzyme marker analyses. Guanosine triphosphate (GTP) binding assays were carried out on the extract with guanosine 5'[γ-thio] triphosphate, [35S] (GTPγ35S) as the non-hydrolysable substrate. A specific binding was observed and a KD of 0.3 mM was estimated by displacement curves. The characteristic enhancement by Mas 7 was also observed. Two dimensional gel electrophoresis of the GTP-binding test permitted localization of proteins between 20 and 55 kDa which bound specifically to GTPγ35S. Immunodetection was performed in the same experimental conditions with an antibody raised against the conserved consensus sequence of the GTP-binding site of the animal Gα subunit of the heterotrimeric G-Protein. It confirmed the presence of proteins of 41–44 kDa which correspond to those detected with the GTP-binding test.