BACKGROUND: Studies were carried out to characterize several biochemical features of cultured AT-1 cells. METHODS AND RESULTS: These cells were obtained from a transplantable atrial cardiomyocyte tumor lineage. Reverse transcriptase-polymerase chain reaction-based analyses demonstrated that the pattern of gene expression of cultured AT-1 cells was similar to that of adult atrial myocytes. AT-1 cells expressed atrial natriuretic factor (ANF), alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. Radioimmunoassays verified that the cells synthesized, stored, and secreted ANF. Through size-exclusion, reversed-phase, and carboxymethyl-ion-exchange high-performance liquid chromatography, it was shown that cultured AT-1 cells stored ANF as pro-ANF (ANF-[1-126]), which was cosecretionally processed quantitatively to ANF-(1-98) and the bioactive 28-amino-acid ANF-(99-126). In addition, cultured AT-1 cells secreted ANF at almost a sixfold greater rate in response to endothelin-1, a potent secretagogue of ANF. KCl, metenkephalinamide, isoproterenol, phenylephrine, and 12-O-tetradecanoyl-phorbol-13-acetate also stimulated ANF release. CONCLUSIONS: These studies, in combination with previous findings, demonstrated that cultured AT-1 cells, while maintaining the ability to proliferate, have retained functional, biochemical, and ultrastructural features that are characteristic of adult atrial myocytes.