The Popeye domain containing (Popdc) genes encode membrane proteins with a preferential expression in striated muscle. In vertebrates three genes are present in the genome of which Popdc2 expression is largely restricted to the heart, while Popdc1 and -3 are more widely expressed. In order to analyze their function we have created Popdc1 and Popdc2 null mutant mice by creating lacZ knock-in mutations. Both mouse strains displayed normal viability during postnatal life. Detailed analysis of the LacZ expression pattern of both mutants revealed a strong expression in the cardiac conduction system in the postnatal heart. Electrophysiological analysis of both mutants revealed conduction abnormalities. Popdc1 mice displayed a sinus bradycardia and a retarded AV node conductivity, which became pronounced after beta-adrenergic stimulation. Popdc2 developed a load-induced sinus bradycardia after subjecting mutant mice to an exercise test or to prolonged mental stress by airjet. The stress-induced bradycardia in Popdc2 mice was due to a sinus bradycardia and not due to prolonged AV conduction or AV nodal block. Interestingly, the bradycardic phenotype developed over time and was not present at 2 month of age, at 5.5 month significant differences between mutants and wildtype animals was observed in case of the exercise test, while at 9 month both exercise and mental stress elicited a bradycardic response. Histological analysis revealed structural defects in sinus node morphology. Hcn4 expression was reduced in the mutant and sinus node cells appeared to be progressively substituted by working myocardium. In support of this view the sinus node of Popdc2 mutant displayed co-expression of Cx30.2 and Cx43. Knock-down of popdc2 in zebrafish embryos revealed an essential function of popdc2 for cardiac and skeletal muscle development. Injected embryos displayed a disorganized tail musculature and a dysmorphic heart. At low morpholino concentrations some of the morphant’s hearts displayed irregular cardiac contractions, a phenotype that is reminiscent of the conduction abnormalities observed in the mutant mouse heart. In conclusion the Popdc family plays an important role for heart and muscle development in zebrafish and is essential for the conduction system function in the adult mouse heart.