Apetala2/ethylene-responsive factor (AP2/ERF) proteins are AP2 domain-containing transcription factors and form the second largest transcription factor family in plants. Biological functions of many of these AP2 proteins are still unknown. Here, we report the characterisation of a novel member of the AP2/ERF superfamily, dehydration-responsive factor 1 (HvDRF1) from barley, and its role in abscisic acid (ABA)-mediated gene regulation. The expression of HvDRF1 was upregulated in barley leaves and roots under drought, salt or ABA treatment, and in embryos during seed maturation. Three forms of HvDRF1 transcripts were produced through alternative splicing, two of which encoded AP2 proteins. This alternative splicing pattern was also observed in a wheat homologue gene, TaDRF1. Both of HvDRF1 AP2 proteins acted as transcriptional activators, capable of activating the promoter activity of an ABA-inducible HVA1s in barley. In vitro DNA-binding analysis using synthetic oligonucleotides revealed that HvDRF1 AP2 protein bound preferably to a CT-rich element (T(T/A)ACCGCCTT). HvDRF1 activity on the activation of HVA1s expression in barley leaves was markedly enhanced by HvABI5 (a bZIP transcription factor), ABA or drought treatment. These results indicate that the HvDRF1 transcriptional activator co-operates with other ABA-responsive factors in the upregulation of stress gene expression through an ABA-dependent pathway.