The Popeye domain containing gene family consists of three genes Popdc1, Popdc2, and Popdc3. Popdc genes encode membrane proteins having a short glycosylated amino terminus, followed by three transmembrane domains, and a long cytoplasmic portion. The cytoplasmic-localized Popeye domain acts as a cyclic nucleotide-binding domain. Popdc genes are abundantly expressed in heart and skeletal muscle and are required for skeletal muscle regeneration. In addition, Popdc1 and Popdc2 null mutants display a stress-induced bradycardia. Here we have studied the subcellular localization of Popdc proteins after cell transfection and with the help of isoform-specific antibodies. Transfection of full-length protein into Cos7 cells revealed intracellular and plasma membrane localization, however, transfection of a deletion construct encoding only the cytoplasmic domain revealed nuclear localization. Isoform-specific antibodies against each of the three family members were employed to study the endogenous protein distribution in adult cardiac and skeletal muscle cells. In adult cardiac myocytes Popdc1 was found at the plasma membrane and in T-tubules. Popdc2 and Popdc3 showed a similar protein distribution but in addition also revealed nuclear localization. In adult skeletal muscle cells Popdc1 was present in the plasma membrane and in T-tubules, and a similar distribution was seen for Popdc2. Nuclear localization of Popdc1 protein was observed in satellite cells and in C2C12 cells. Interestingly, nuclear localization was differentiation-dependent and was not found in myotubes. This observation was extended to Popdc2 and Popdc3, which revealed a similar protein distribution, however, in contrast to Popdc1, Popdc2 persisted for longer periods in the nucleus and Popdc3 was present in the nucleus even in fully differentiated myotubes. The role of Popdc1 in muscle differentiation was studied in satellite cells isolated from wild type and Popdc1 null mutants. We found that wild type and mutant satellite cells differed in their proliferative behavior, which in part might depend on nuclear localization of Popdc1 in these cells.Taken together, we found that Popdc proteins, which are transmembrane proteins, are also localized in the nucleus of adult cardiac myocytes as well as muscle satellite cells. This novel feature of nuclear localization may be of significance for the function of this protein family in muscle tissues, and may have an impact on skeletal muscle regeneration.