An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.