We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates.