Rhythm disorders and sinoatrial node (SAN) dysfunctions often result in the implantation of electronic pacemakers. The progress in stem cell research has opened the possibility to regenerate the cardiac pacemaker tissue. Data in the literature indicate that CD166 is specifically but transiently expressed in the developing heart tube and in the prospective SAN and can be used to enrich human embryonic stem cells (ESCs) in cardiomyocytes. Here we used CD166 as a marker to isolate SAN progenitors from mouse ESCs. Mouse ESCs differentiation was induced by embryoid bodies (EBs) formation. EBs were dissociated, cells labeled with an anti-CD166 antibody and sorted. Quantitative RT-PCR, immunofluorescence and electrophysiological analyses were carried out to characterize CD166+ cells. We found that CD166+ cells derived from 8 days-old EBs express high levels of various cardiac genes. Specifically they express genes involved in SAN development (Tbx18, Isl-1, Shox2) and SAN function (HCN4, HCN1, CACNA1D) while express low levels of ventricular genes (Cx43, Kv4.2, Nkx2-5). CD166+ cells beat spontaneously and their rate can be increased (+57%) by the adrenergic agonists, isoproterenol (1mM) and decreased (-23%) by acetylcholine (0.1mM). they also express the HCN1 and HCN4 pacemaker channels accounting for an If current resembling that of the native SAN. In conclusion, our data demonstrate that the CD166 can be used to isolate a homogeneous population of sinoatrial precursors from ESCs.