AbstractN‐RAP gene expression and N‐RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase‐polymerase chain reaction and immunofluorescence. N‐RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5–10.5 heart tissue, N‐RAP protein was primarily associated with developing premyofibril structures containing α‐actinin, as well as with the Z‐lines and M‐lines of more‐mature myofibrils. In contrast, N‐cadherin was concentrated in patches at the periphery of the cardiomyocytes. N‐RAP labeling markedly increased between E10.5 and E16.5; almost all of the up‐regulated N‐RAP was associated with intercalated disk structures, and the proportion of mature sarcomeres containing N‐RAP decreased. In adult hearts, specific N‐RAP staining was only observed at the intercalated disks and was not found in the sarcomeres. The results are consistent with N‐RAP functioning as a catalytic scaffolding molecule, with low levels of the scaffold being sufficient to repetitively catalyze key steps in myofibril assembly. Developmental Dynamics 233:201–212, 2005. © 2005 Wiley‐Liss, Inc.