In the early embryonic heart, endothelial cells in atrioventricular (AV) and outflow tract (OT) regions are transformed into the invasive mesenchymal cells that form endocardial cushion tissue (endothelial-mesenchymal transformation). It has been reported that bone morphogenetic proteins (BMPs) are transcribed in the AV and OT regions of the embryonic mouse heart. We previously reported that transforming growth factor beta 3 (TGFbeta3) triggers the initial phenotypic changes seen in endothelial-mesenchymal transformation. We cloned BMP2 from embryonic chick hearts and examined its functional role during endocardial cushion tissue formation. In situ hybridization showed BMP2 transcripts in the myocardium of the AV and OT regions, but not in endothelial/mesenchymal cells. Antisense oligodeoxynucleotides to BMP2 inhibited mesenchyme formation in AV endocardium cocultured with associated myocardium. This inhibitory effect was reversed by the addition of recombinant BMP2. In cultured AV endothelial monolayers, recombinant BMP2 did not induce any cellular phenotypic changes characteristic of endothelial-mesenchymal transformation. However, BMP2 enhanced the TGFbeta-induced initial phenotypic changes associated with endothelial-mesenchymal transformation. These results suggest that BMP2 1) plays an important role in the formation of endocardial cushion tissue and 2) acts synergistically with TGFbeta3 in the regulation of this developmental event.