Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists concerning the expression patterns of atrial (AMHC), ventricular (VMHC), and cardiac myosin heavy-chain (CMHC) during development in the heart. In this study, we describe that probe length, probe concentration, and staining time in the non-radioactive in situ hybridization procedure seriously influence the observed pattern of MHC expression and the subsequent interpretation, explaining the divergent opinions in the field. Using a variety of external and internal controls for the in situ hybridization procedure, we demonstrate that both AMHC and VMHC are expressed throughout the entire heart tube during early development. During subsequent development, VMHC becomes restricted to the ventricles, whereas AMHC remains expressed in the atria, and, at substantially lower levels, is detected in the ventricles. These results are discussed in the context of methodological constraints of demonstrating patterns of gene expression. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.