In adult cardiac cells the established mechanism of excitation-contraction coupling is by calcium-induced calcium release (CICR) mediated by L-type Ca(2+) channels. However, in neonate cardiomyocytes, a CICR modality involving reverse mode Na(+)-Ca(2+) exchanger (NCX) activity predominates. This has been hypothesized to be due, in part, to the high expression levels of NCX in the neonate heart which drop several fold during ontogeny. Very little is known about the nature of NCX distribution within the cardiomyocyte and how this might change with development given the significant differences in gene expression. We investigated the spatial arrangements of NCX in developing rabbit ventricular myocytes with traditional as well as novel image processing and analysis techniques. Using image segmentation, colocalization analysis was conducted at the whole cell, compartmental (cell periphery and cell interior) and object levels. Because NCX has been suggested to colocalize with caveolin-3 (cav-3) and perhaps form a signaling unit within caveolae, the spatial relationship of NCX relative to cav-3 was also examined in detail. NCX and cav-3 objects were found to be isolated islands of lit voxels that are present after thresholding. These objects were categorized into non-colocalized (0%), lowly colocalized (<50%) and highly colocalized (>50%) subpopulations in both the interior and peripheral compartments. Our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete objects and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 objects are organized with a longitudinal and lateral periodicity of about 1mum and that NCX and cav-3 cluster appear to be mutually exclusive on the cell periphery. We conclude that despite the very significant decrease in NCX expression with maturation, qualitatively there were no differences in NCX surface distribution or in the spatial relationship to caveolin 3.