The formation of cardiac cushion tissue, which ultimately contributes to formation of the valves and septa, is dependent on the regional activation of cardiac endothelial cells to undergo an epithelial-mesenchymal transition. This endothelial transition was correlated with activin โคA mRNA expression by Northern and in situ hybridization in both a temporal and spatial manner in developing mouse embryos. Activin โคA was the only subunit of the inhibin family detected during the initial phase of endothelial cell transition; activin โคB was detected at later stages, and inhibin โฃ was not detectable in the heart. An in vitro assay that has been used to study mesenchymal cell formation in chick was modified for use with mammalian embryos. Conditioned media from embryonic mouse cardiocyte cultures was shown to substitute for the endogenous inductive signal in these assays. The presence of activin โคA was demonstrated by Western blot analysis of the cardiocyte conditioned media (CCM). Modified antisense oligonucleotides to activin โคA inhibited the endothelial-mesenchymal transition in the assay system, which was not affected by control oligonucleotides. Adapting the avian culture system for use with mice enabled the use of tissue from mice with a null allele for activin โคA. CCM produced from embryos homozygous for the mutant โคA allele did not contain activin โคA and was used in in vitro assays. CCM lacking activin โคA produced fewer mesenchymal cells from cardiac endothelial monolayers than CCM with activin โคA. Localized expression of activin โคA in the embryonic heart indicates a possible role in the endothelial-mesenchymal transition. Bioassays in which activin โคA expression is blocked or activin โคA is absent from the media indicate that activin โคA promotes the formation of mesenchymal cells in the endothelial cushions, which are required for normal septation.