Primary cultures of neonatal rat atrial and ventricular cardiomyocytes were used to investigate the expression of peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme required for the production of alpha-amidated neuroendocrine peptides. The use of assays for the individual enzymes, peptidylglycine alpha-amidating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), demonstrated that the levels of expression observed in vitro approximated those observed in vivo. Both in vivo and in vitro, atrial and ventricular PAL activity greatly exceeded PHM activity. Atrial and ventricular cardiomyocytes secreted PHM and PAL activity at a constant rate throughout the culture period. Immunofluorescence studies localized PAM proteins to the perinuclear region, with intense punctate staining. Both in vivo and in vitro, PAM mRNAs encoding integral membrane proteins predominated throughout the neonatal period, with PAM-1 mRNA becoming more prevalent after the first week in culture. Although PAM-2 mRNA decreased in prevalence in vivo at the time when PAM-1 expression increased, levels of PAM-2 mRNA remained elevated throughout 2 weeks in vitro. Western blot analysis demonstrated intact PAM-1 and PAM-2 proteins in atrial cultures, with the prevalence of PAM-1 increasing in older cultures. Atrial cardiomyocytes secreted only bifunctional PAM proteins. Many of the features of PAM expression, processing, and storage that are unique to cardiomyocytes as opposed to endocrine cells are faithfully replicated by primary atrial and ventricular cultures.