In newborn (NB) heart, the contribution of ryanodine receptors (RyRs) to the excitation-contraction coupling (ECC) is minimal, despite their abundance and their adult (AD)-like single-channel behavior. We hypothesized that, although RyRs form operational release sites in the cell center they do not contribute to ECC in NB, because the plasmalemmal Ca²⁺ signal in the surface does not activate them. Thus, Ca²⁺ sparks and their distribution were determined in NB and correlated with the formation of dyads and transverse tubules (TTs). Central and peripheral sparks were defined/compared in single myocytes from AD and NB rats (D0-D9). Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyadic formation, while the TTs development was studied with fluorescence imaging and electron microscopy (EM). During the first week, the fraction of central sparks increased from 45 to >70%. Immunolocalization of D7 myocytes, revealed abundant central dyads; however, plasmalemma fluorescence labeling failed to show internal TTs. This could represent central release sites associated with DHPR-containing TTs that are unconnected to the surface. Ferritin was used as an electron dense tracer to define in EM, continuity with the extracellular space of central TTs. These studies indicated that at D2, 40% of dyadic central TTs were open, while at D8 this fraction was 66%. Our results suggest that TTs form by active invagination of the plasmalemma and by simultaneous genesis of internal-unconnected TT/SR dyads that eventually connect to surface. This results would explain the developmental quiescence of some RyRs during ECC. (Supported by NIH-HL071741, NIH-HL62571, AHA-9950382N).