Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.