Plant bZIP proteins exhibit a relaxed DNA-binding specificity for DNA sequence motifs containing an ACGT core. Gel mobility shift experiments employing ten different recombinant plant bZIP proteins demonstrated that nucleotides flanking the ACGT core affected binding specificity and identified three different types of ACGT elements: G-box, CACGTG; C-box, GACGTC; and A-box, TACGTA, motifs. These ten different bZIP proteins could be categorized into three groups according to their qualitative and quantitative specificity for G-box and C-box elements. Dissociation constant values (Kd values) of these bZIP proteins for high affinity G-box and C-box elements and reciprocal competition gel mobility shift assays confirmed our classification scheme. Group 1 proteins exhibit a stronger binding affinity for G-box elements, group 2 proteins bind both G-box and C-box motifs with comparable binding affinity, whereas the group 3 proteins display a stronger binding affinity for C-box oligonucleotides. Studies using a panel of G-box and C-box oligonucleotides differing in their flanking sequences identified high affinity binding sites. All ten plant bZIP proteins examined, except TGA1a, exhibited type A G-box binding activity preferring class I G-box elements. In contrast to the situation observed for G-box elements, C-box motifs displayed a very much more stringent flanking nucleotide requirement for binding activity. Protein/DNA binding experiments using scanning mutants of a high affinity G-box element and G-box/C-box hybrid elements demonstrated that bZIP protein binding activity depends upon the affinity of protein dimer subunits for ACGT half-sites. Information provided by our systematic analysis of plant bZIP DNA binding specificity can be used to identify high affinity binding sites for the plant bZIP proteins studied here. Assuming that only high affinity bZIP binding sites are likely to function in vivo, identification of these sites will allow us to predict which genes are activated by a particular bZIP protein.