The G-box is a cis-acting DNA sequence present in several plant promoters that are regulated by diverse signals such as UV irradiation, anaerobiosis, abscissic acid and light. Several basic/leucine zipper (bZIP) proteins from different plant species have been identified as high affinity G-box binding proteins. Although their capability to enhance transcription has been demonstrated, their precise function in transcriptional activation is still unknown. We have isolated three cDNAs from young tomato fruit that encode bZIP G-box binding proteins (GBF4, GBF9 and GBF12). They bind to the G-box sequence in the tomato rbcS1, rbcS2 and rbcS3A promoters. GBF9 binding resulted in a DNase I footprint identical to that obtained with tomato nuclear extract and different from the DNase I protection obtained with GBF4 and GBF12. The mRNAs of all three GBFs were most abundant in tomato fruit and seeds, moderately abundant in root and least abundant in leaves. Protein sequences outside of the bZIP domains were compared with the known GBFs from other plants and seven conserved motifs of seven to 35 amino acids length have been identified. Based on the presence of these motifs, three classes of GBFs can be defined that are conserved among plant species. GBF9, the predominantly expressed tomato GBF, is the first member of its class isolated from dicot plants. Three conserved motifs from two of the classes are highly hydrophilic and are predicted to be exposed on the surface of the proteins. These motifs likely define novel interactive domains in the different classes of GBFs that could provide a new tool to determine how distinct regulatory signals are transmitted through GBFs to activate transcription.