This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.