Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.