The yeast one-hybrid (Y1H) system is a powerful tool for the identification and isolation of cDNAs of transcription factors using promoter segments or regulatory elements as baits. Here we propose an adaptation of the Y1H system for identification and cloning of transcription factors using Matchmaker (Clontech) Y2H cDNA libraries. The method is a modification of the standard one-hybrid screening protocol, utilising a mating step to introduce the library and reporter constructs into the same cell. This extends the compatibility of Matchmaker cDNA libraries from yeast two-hybrid screens to one-hybrid screens. Libraries were successfully prepared from wheat, barley and maize grain, spike, leaf and root tissues from plants subjected to several environmental stresses. Using this method, we have isolated more than 50 cDNAs encoding transcriptional factors from several different families.