Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for differentiation of early mesoderm and cardiomyocytes. Addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on Day 6 of expansion and then plateaued until Day 20. In real-time PCR analysis, cardiac development-related genes Mesp1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. Activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8, and by nuclear localization of phospho-Smad1/5/8 and Smad4. Addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. Cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promote cardiomyocyte induction from human ES cells.