In order to compare within the same cell the various degrees of specificity of myosin alkali light chain (MLC) isoproteins sorting to sarcomeres, a competition assay was established using double epitope tagging. Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) protein epitope were co-expressed in cultured cardiomyocytes from adult and neonatal rat ventricles. Expressed isoproteins were detected by means of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy. The sorting specificity of MLC isoforms to sarcomeric sites was shown to increase in the order MLC3nm, to ML1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developmental expression. Expressed fast skeletal muscle isoforms (MLC1f and MLC3f) were always localized at the A-bands of myofibrils, while nonmuscle type (MLC3nm) was distributed throughout the cytoplasm. The slow skeletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it was co-expressed with MLC3nm, but it was distributed throughout the cytoplasm when expressed in combination with MLC1f, MLC3f or the slow skeletal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it was also distributed to the cytoplasm if co-expressed with MLC1f or MLC3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorting pattern.