GT-2 is a plant transcriptional activator that contains two separate, but similar, trihelix DNA-binding domains. GT-1 is similar to GT-2, although it contains only one of such domains. cDNAs that encode GT-2 were isolated from rice (OS-GT2) and Arabidopsis (AT-GT2). Evidence is presented for the existence of an Arabidopsis gene family that is structurally related to AT-GT2. Two members of this GT2-like family, AT-GTL1 and AT-GTL2, have been isolated and characterized. Their sequences suggest that they evolved by a recent gene duplication event. Both AT-GT2 and AT-GTL genes contain an intron in the amino-terminal trihelix motif, indicating that this DNA-binding domain resulted from exon shuffling. RNA gel blot analysis using AT-GTL1 as a probe revealed four transcripts in the aerial part of the plant. All mRNA levels were significantly higher in siliques, suggesting that this gene family may function in fruit and/or seed development. To date, DNA-binding proteins characterized by the trihelix motif have been described only in plants, and may therefore be involved in plant-specific processes. Our results show that in Arabidopsis thaliana, the trihelix motif is not restricted to the GT-1 and GT-2 DNA-binding proteins.