Cardiac-specific deletion of the receptor IA of bone morphogenetic protein (BMP) (ALK3) by Cre recombinase driven under the α-MHC promoter is lethal in mid-gestation with defects in the interventricular septum [ventricular septum defect (VSD)]. Analysis of expression of the ALK3 downstream genes is important to identify the signaling pathway for interventricular septum development. The mRNA expression level of a control group was compared with that of a test group. ALK3 downstream genes were screened using polymerase chain reaction (PCR)-select cDNA subtraction and microarray. It was found that the mice with an ALK3 knockout gene produced a VSD. The expression of some genes such as platelet-activating factor acetylhydrolase (PAF) and Pax-8 was down-regulated in the test group. Pax-8 gene expression was down-regulated by 7.1 times in the test group and expressed specifically in the 11.5-d embryonic (E11.5) heart. Furthermore, the expression of the protein-tyrosine kinase of the focal adhesion kinase subfamily (PTK) and β subtype protein 14-3-3 was up-regulated in the test group. PTK gene expression was up-regulated by 3.7 times in the test group. These data provided support that the ALK3 gene plays an important role during heart development. The PAF and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and β subtype protein 14-3-3 might be regulatory factors in this pathway.