This paper describes the cDNA cloning, genomic organization, and expression of the human RTN2 gene on chromosome 19q13.3, which was recognized by virtue of its high similarity with the human RTN1 (formerly called NSP) gene on chromosome 14q21-q22. In a region of about 12 kb in total, 11 RTN2 exons could be identified. Like the RTN1 gene, the RTN2 gene is transcribed into different mRNA variants. Two have a size of about 2.3 kb, and a third has a size of about 1. 3 kb. The two 2.3-kb transcripts differ because of alternative splicing of exon 5. Transcription of the 1.3-kb transcript starts presumably from an internal promoter within exon 5. The three mRNAs encode three different proteins, RTN2-A (545 aa), RTN2-B (472 aa), and RTN2-C (205 aa), which share a common carboxy-terminal segment of 201 aa. In this common segment, the homology with the RTN1 proteins, with yet unknown function, is found. Two hydrophobic subregions are present, which are thought to be responsible for the association of the RTN1 and RTN2 proteins with the endoplasmic reticulum. The amino-terminal regions of the RTN2-A and RTN2-B proteins are rich in negatively charged residues and in proline and serine residues and contain multiple potential phosphorylation sites. Analysis of the expression of the RTN2 gene shows differential expression in human tissues with a strikingly high expression of the 1.3-kb transcript in skeletal muscle.