The cells that form the cardiac conduction system (CCS) are recruited from embryonic cardiomyocytes. Endothelin‐1 (ET‐1) and Neuregulin‐1 (NRG‐1) have been associated with this transition in the avian and murine systems, respectively. We established murine embryonic cardiomyocyte cultures induced or not with ET‐1 and/or NRG‐1 to compare the expression of cardiomyocyte and CCS‐specific genes. Semiquantitative reverse transcription‐polymerase chain reaction analysis showed that cardiomyogenesis and CCS‐specific markers, such as Nkx2.5, GATA4, Irx4, Connexin 40, Connexin 45, HF‐1b, and MinK, were up‐regulated in the presence of either growth factor. Additionally, immunofluorescence analysis demonstrated that ET‐1 or NRG‐1 increased the number of cells expressing the Purkinje fiber‐specific marker Connexin 40 in induced cultures but did not selectively increase their proliferation rate. Interestingly, additive effects were not observed in ET‐1 and NRG‐1 combination treatments. Among other possibilities, this observation suggests that these factors may interact to promote the differentiation of the murine CCS. Developmental Dynamics 233:20–28, 2005. © 2005 Wiley‐Liss, Inc.