Nebulin-related anchoring protein (N-RAP) is a 185 ku actin-binding LIM protein recently discovered in skeletal and cardiac muscle tissues in mouse. It is proposed that N-RAP serves as a link between the terminal actin of the myofibril and the protein complexes at the cell membrane and thus as an organizing center in the initial phase of myofibril assembly. But in human, the sequence and function of N-RAP remain unknown. By using bioinformatics tools, the full length of human N-RAP cDNA, which contains a 5 088 bp ORF, encodes a protein of 1 695 amino acid residues, is successfully cloned. Human N-RAP is mapped to the genomic region between HABP2 and CASP7 at chromosome 10q25～q26, consisting of 41 exons and 40 introns. Homology searches with the deduced 1 695 amino acid protein sequence reveal human N-RAP shares 88% similarity with mice N-RAP, 63% with human Nebulin and 59% with mice Nebulin. Corresponding EST sequences are found in muscle, heart, spinal cord and prostate tissue. The predicted protein contains LIM domain (5～57), which binds two zinc ions, does not bind DNA, seems to act as interface for protein-protein interaction, and Nebulin repeats, tandem arrays of which are known to bind actin. RT-PCR reveals human N-RAP is expressed in adult muscle, heart and brain tissue, not in bone marrow. In addition, subcellular location study shows human N-RAP is expressed in cytoplasm. These results demonstrate that just like mice N-RAP, human N-RAP is proposed to be crucial for myofibrillogenesis.