Cardiac pacemaker cells residing in the sinoatrial node (SAN), an important component of the cardiac conduction system (CCS), are responsible for setting the pace of the heartbeat and therefore are crucial for maintaining a proper heart rhythm. However, the molecular control of pacemaker development in mammals and especially in zebrafish is unclear. With the increasing popularity of the zebrafish as a model for studies of cardiac physiology and disease, a better characterization of the cardiac pacemaker cells is necessary. By transposon-mediated insertional transgenesis, hundreds of enhancer trap (ET) lines that express EGFP in distinct anatomical localization have been generated. Two of them, termed SA lines, have distinct EGFP expression at the venous pole of the heart, which can be observed from as early as 36 hours post fertilization. These EGFP-positive cells were characterized at different developmental stages and were confirmed by immunostaining and marker gene expression to be cardiac pacemaker cells. The genomic transposon integration site in the SA lines was determined to be on a defined region on chromosome 14. By whole-mount in situ hybridization, the fibroblast growth factor homologous 2 (fhf2) gene was identified to be the gene whose expression pattern was recapitulated by the SA lines. While initially only the sino-atrial junction displayed EGFP expression, a second expression domain in the AV canal became visible in older embryos. Both expression domains were also present in the adult zebrafish heart. The loss-of-function phenotype of fhf2 was studied by morpholino-mediated knockdown, which resulted in a malfunctioning heart developing pericardial oedema and blood accumulation at the venous pole. In order to test the importance of SAN pacemaker cells in the zebrafish heart, we analysed the electrophysiological phenotype of a mutant which lacked pacemaker cells based on the absence of fhf2 and other pacemaker gene expression. The mutant heart was found to display a severe bradyarrhythmia with extensive pauses indicating the importance of cardiac pacemaker cells in proper heart function. In summary, we describe the first characterization of the cardiac pacemaker cells of the zebrafish and the functional consequences of their absence to the functioning of the heart.