The cardiogenic capacity of embryonic stem (ES) cells has been well-investigated. However, little is known about the development of adrenoceptor (AR) systems during the process of ES cell differentiation, which are critically important in cardiac physiology and pharmacology. In this present study, we investigated the expression profile of adrenoceptor subtypes, β-adrenergic modulation of muscarinic receptors and adrenoceptor-related signaling in cardiomyocytes derived from ES cells (ESCMs).Reverse transcription-polymerase chain reaction revealed that undifferentiated mouse ES cells expressed α1A-, α1B-, α1D- and β2-AR mRNA. However, β1-AR was only expressed after vitamin C induction. The expressions of α1A-, α1D- and β1-ARs increased significantly while α1B- and β2-ARs showed no significant change during the differentiation process. Furthermore, we detected the expression of tyrosine hydroxylase. Both α1-AR and β-AR could activate extracellular responsive kinase in ESCMs. Isoprenaline could inhibit the expression of M2 muscarinic receptor protein. CGP20712A, a β1-AR antagonist, up-regulated the expression of M2 muscarinic receptor while ICI118551, a β2-AR antagonist, showed no effect.These results indicated that functional adrenoceptors and tyrosine hydroxylase, a critical enzyme in catecholamine biosynthesis, were differentially expressed in ESCMs. Adrenoceptor-related signaling pathways and β-adrenergic modulation of muscarinic receptors were established during differentiation.