MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the 'ABC model' of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as Bsister (Bs) genes. Here, we report the isolation and characterization of a MADS-box Bs member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis Bs mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between Bs-C-E and Bs-D-E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development. © 2006 The Authors.