Summary The screening of enhancer detector lines in Arabidopsis thaliana has identified genes that are specifically expressed in the sporophytic tissue of the ovule. One such gene is the MADS-domain transcription factor AGAMOUS-LIKE6 (AGL6), which is expressed asymmetrically in the endothelial layer of the ovule, adjacent to the developing haploid female gametophyte. Transcription of AGL6 is regulated at multiple stages of development by enhancer and silencer elements located in both the upstream regulatory region and the large first intron. These include a bipartite enhancer, which requires elements in both the upstream regulatory region and the first intron, active in the endothelium. Transcription of the AGL13 locus, which encodes the other member of the AGL6 subfamily in Arabidopsis, is also regulated by elements located in the upstream regulatory region and in the first intron. There is, however, no overlapping expression of AGL6 and AGL13 except in the chalaza of the developing ovule, as was shown using a dual gene reporter system. Phylogenetic shadowing of the first intron of AGL6 and AGL13 homologs from other Brassicaceae identified four regions of conservation that probably contain the binding sites of transcriptional regulators, three of which are conserved outside Brassicaceae. Further phylogenetic analysis using the protein-encoding domains of AGL6 and AGL13 revealed that the MADS DNA-binding domain shows considerable divergence. Together, these results suggest that AGL6 and AGL13 show signs of subfunctionalization, with divergent expression patterns, regulatory sequences and possibly functions. Journal compilation © 2009 Blackwell Publishing Ltd.